Novel immunotherapy against several tumors, such as lung cancer, including nsclc

ABSTRACT

The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated cytotoxic T cell (CTL) peptide epitopes, alone or in combination with other tumor-associated peptides that serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses. The present invention relates to more than 70 novel peptide sequences and their variants derived from HLA class I and HLA class II molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses.

The present invention relates to peptides, nucleic acids and cells foruse in immunotherapeutic methods. In particular, the present inventionrelates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated cytotoxic T cell (CTL) peptideepitopes, alone or in combination with other tumor-associated peptidesthat serve as active pharmaceutical ingredients of vaccine compositionsthat stimulate anti-tumor immune responses. The present inventionrelates to 67 novel peptide sequences and their variants derived fromHLA class I and HLA class II molecules of human tumor cells that can beused in vaccine compositions for eliciting anti-tumor immune responses.

BACKGROUND OF THE INVENTION

Lung cancer accounts for the most cancer-related deaths in both men andwomen. Worldwide, lung cancer is the most common cancer in terms of bothincidence and mortality. In 2008, there were 1.61 million new cases, and1.38 million deaths due to lung cancer. The highest rates are in Europeand North America.

Since 1987, more women have died each year from lung cancer than frombreast cancer. Death rates have continued to decline significantly inmen from 1991-2003 by about 1.9% per year.

Female lung cancer death rates are approaching a plateau aftercontinuously increasing for several decades. These trends in lung cancermortality reflect the decrease in smoking rates over the past 30 years.

An estimated 230,000 new cases of lung cancer and 160,000 deaths due tolung cancer are expected in 2013 in the USA according to the nationalcancer institute (NCI).

Lung cancer is classified clinically as small cell (13%, SCLC) ornon-small cell (87%, NSCLC) for the purposes of treatment. Prognosis isgenerally poor. Of all people with lung cancer, 15% survive for fiveyears after diagnosis. Stage is often advanced at the time of diagnosis.At presentation, 30-40% of cases of NSCLC are stage IV, and 60% of SCLCare stage IV.

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab(AVASTIN®) and erlotinib (TARCEVA®). For localized cancers, surgery isusually the treatment of choice. Recent studies indicate that survivalwith early-stage, non-small cell lung cancer is improved by chemotherapyfollowing surgery. Because the disease has usually spread by the time itis discovered, radiation therapy and chemotherapy are often used,sometimes in combination with surgery. Chemotherapy alone or combinedwith radiation is the usual treatment of choice for small cell lungcancer; on this regimen, a large percentage of patients experienceremission, which is long lasting in some cases.

The 1-year relative survival for lung cancer has slightly increased from37% in 1975-1979 to 42% in 2002, largely due to improvements in surgicaltechniques and combined therapies. However, the 5-year survival rate forall stages combined is only 16%. The survival rate is 49% for casesdetected when the disease is still localized; however, only 16% of lungcancers are diagnosed at this early stage.

Despite the above, there remains a need for new efficacious and safetreatment option for cancers such as lung cancer, in particularnon-small-cell lung cancer (NSCLC), gastric cancers and brain tumors ofdifferent phenotypes which improve the well-being of the patients by notusing excessive chemotherapeutic agents or other agents that may lead tosevere side effects.

The present invention employs peptides that stimulate the immune systemof the patient and act as anti-tumor-agents in a non-invasive fashion.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup of SEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ ID No.84, and SEQ ID No. 92 or a variant sequence thereof which is at least80%, preferably at least 90%, homologous (preferably at least 80% or atleast 90% identical) to SEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No. 76to SEQ ID No. 84, and SEQ ID No. 92, wherein said variant induces Tcells cross-reacting with said peptide, or a pharmaceutical acceptablesalt thereof, wherein said peptide is not a full-length polypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the group of SEQID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to, and SEQ ID No. 84, andSEQ ID No. 92 or a variant thereof, which is at least 80%, preferably atleast 90%, homologous (preferably at least 80% or at least 90%identical) to SEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ IDNo. 84, and SEQ ID No. 92, wherein said peptide or variant thereof hasan overall length for SEQ ID No. 1 to SEQ ID No. 65 and SEQ ID 78 to SEQID No. 84 and SEQ ID No. 92 of between 8 and 100, preferably between 8and 30, and most preferred of between 8 and 14 amino acids, and for SEQID No. 76 and 77 of between 12 and 100, preferably between 12 and 30,and most preferred of between 12 to 18 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NO, and the prospective sourceproteins for these peptides. All peptides in Tables 1a, 1b and 1c bindto the HLA A*02 allele, peptides in Table 1d bind to HLA-DR alleles. Thepeptides in table 1c are further useful in the diagnosis and/ortreatment of gastric cancer and or glioblastoma.

The class II peptides in table 1d are further useful in the diagnosisand/or treatment of gastric cancer and other cancers over-expressing orover-presenting MMP12 or POSTN.

Thus, the present invention relates in particular to a peptide of thepresent invention comprising a sequence according to SEQ ID No. 76 or avariant thereof, which is at least 80%, preferably at least 90%,homologous (preferably at least 80% or at least 90% identical) to SEQ IDNo. 76, wherein said peptide or variant thereof has an overall length ofbetween 12 and 100, preferably between 12 and 30, and most preferred ofbetween 12 to 18 amino acids. The present invention relates inparticular to a peptide of the present invention consisting of thesequence according to SEQ ID No. 76.

Also, the present invention relates in particular to a peptide of thepresent invention comprising a sequence according to SEQ ID No. 77 or avariant thereof, which is at least 80%, preferably at least 90%,homologous (preferably at least 80% or at least 90% identical) to SEQ IDNo. 77, wherein said peptide or variant thereof has an overall length ofbetween 12 and 100, preferably between 12 and 30, and most preferred ofbetween 12 to 18 amino acids. The present invention relates inparticular to a peptide of the present invention consisting of thesequence according to SEQ ID No. 77.

TABLE 1a Peptides of the present invention Source SEQ Peptide Pro-ID NO: Code Sequence tein(s)  1 ABCA13-001 ILFEINPKL ABCA13  2 MMP12-003KIQEMQHFL MMP12  3 ABCA13-002 ALDENLHQL ABCA13  4 DST-001 NLIEKSIYL DST 5 MXRA5-001 TLSSIKVEV MXRA5  6 DST-002 KLDETNNTL DST  7 CDK4-001TLWYRAPEV CDK4/CDK6  8 HNRNPH-001 SMSGYDQVL HNRNPH1, HNRNPH2  9TANC2-001 ALMDKEGLTAL TANC2 10 RNF213-001 VLSVVEVTL RNF213 11SLC34A2-001 VLLPVEVATHYL SLC34A2 12 SMYD3-001 SLIEDLILL SMYD3 13 AKR-001YLIHFPVSV AKR1C1, AKR1C2 14 RCN1-001 FQYDHEAFL RCN1,RCN3 15 IL8-001KLAVALLAA IL8 16 P2RY6-001 TVIGFLLPFA P2RY6 17 HUWE1-001 RLLGPSAAADILQLHUWE1 18 VCAN-001 TLYPHTSQV VCAN 19 DROSHA-001 AVVEFLTSV DROSHA 20VCAN-002 ALVDHTPYL VCAN 21 PLEKHA8-001 AILDTLYEV PLEKHA8 22 ACACA-001FLIPIYHQV ACACA 23 ITGA11-001 FLHHLEIEL ITGA11 24 COL12A1-002 FLVDGSWSVCOL12A1 25 ELANE-001 GLYPDAFAPV ELANE 26 SERPINB3-001 KLFGEKTYL SERPINB327 KIF26B-001 TVAEVIQSV KIF26B 28 ANKH-001 SISDVIAQV ANKH 29 NXF1-001RLEEDDGDVAM NXF1 30 RGS4-001 KIYNEFISV RGS4 31 GFPT2-001 AIDGNNHEV GFPT232 CERC-001 KLSWDLIYL CERCAM 33 GALNT2-001 ALLRTVVSV GALNT2 34HNRNPM-001 ALGAGIERM HNRNPM 35 BNC1-001 VLFPNLKTV BNC1 36 FKBP10-001TLVAIVVGV FKBP10 37 FZD-001 VLAPLFVYL FZD1, FZD2, FZD7 38 ATP-001SLHFLILYV ATP2A1, ATP2A2 39 LAMC2-001 RLLDSVSRL LAMC2 40 MXRA5-002GLTDNIHLV MXRA5 41 HSP-002 SILTIEDGIFEV HSPA2, HSPA8 42 VPS13B-001SLWGGDVVL VPS13B 43 CSE1-001 ALFPHLLQPV CSE1L 44 DPYSL4-001 NLLAEIHGVDPYSL4 45 SEC61G-001 AIMGFIGFFV SEC61G 46 ORMDL1-002 TLTNIIHNL ORMDL1 47PCNXL3-001 GVLENIFGV PCNXL3 48 SNRNP20-001 GLIEIISNA SNRNP200

TABLE 1b Additional peptides of the present invention SEQ ID Source NO:Peptide Code Sequence Protein(s) 49 SAMSN1-001 RLLJAAENFL SAMSN1 50STAT2-001 SLLPVDIRQYL STAT2 51 CNOT1-001 YLAPFLRNV CNOT1 52 SHMT2-001ALLERGYSL SHMT2 53 JUNB-001 YLPHAPPFA JUNB 54 TACC3-001 KLVEFDFLGA TACC355 CNOT1-002 SLADFMQEV CNOT1 56 RAD54B-001 SLYKGLLSV RAD54B 57 EEF2-002GLAEDIDKGEV EEF2 58 CCNA2-001 SLIDADPYL CCNA2 59 NET1-001 ILVSWLPRL NET160 C11orf24-001 VVDKTLLLV C11orf24 61 RCC1-001 TLISRLPAV RCC1 62MAGEF1-001 ILFPDIIARA MAGEF1 63 NCAPD2-001 SLAGDVALQQL NCAPD2 64C12orf44-001 AMLAVLHTV C12orf44 65 HERC4-001 KVLEILHRV HERC4

TABLE 1c Additional peptides that are also over-expressed in glioblastoma and/or gastric cancer SEQ ID Source NO:Peptide Code Sequence Protein(s) 66 IGF2BP3-001 KIQEILTQV IGF2BP3 67CDC6-001 ILQDRLNQV CDC6 68 FAP-003 YVYQNNIYL FAP 69 WNT5A-001 AMSSKFFLVWNT5A 70 TPX2-001 KILEDVVGV TPX2 71 HMMR-001 KLLEYIEEI HMMR 72 ADAM8-001KLLTEVHAA ADAM8 73 COL6A3-002 FLLDGSANV COL6A3 74 THY1-001 SLLAQNTSWLLTHY1 75 DIO2-001 ALYDSVILL DIO2

TABLE 1d MHC class II peptides of the invention SEQ ID Source NO:Peptide Code Sequence Protein(s) 76 MMP12-002 INNYTPDMNREDVDYAIR MMP1277 POSTN-002 TNGVIHVVDKLLYPADT POSTN

TABLE 1e Further preferred peptides of the presentinvention with an additional abundance in other cancers SEQ ID SourceNO: Peptide Code Sequence Protein(s) 78 SLI-001 SLYDNQITTV SLIT1, SLIT279 TLX3-001 SLAPAGVIRV TLX3 80 CEP192-001 SLFGNSGILENV CEP192 81ANKS1A-001 ALYGRLEVV ANKS1A 82 CEP250-002 ALWEKNTHL CEP250 83 MDN1-001ALANQKLYSV MDN1 84 OLFM1-001 ILMGTELTQV OLFM1 92 NEFH-001 HLLEDIAHV NEFH

TABLE 1f Further peptides of the present invention withan additional abundance in other cancers SEQ ID Source NO: Peptide CodeSequence Protein(s) 85 BUB1B-001 KIVDFSYSV BUB1B 86 PI4KA-001AMATESILHFA PI4KA 87 AURKB-001 RVLPPSALQSV AURKB 88 SLC3A2-001SLLESNKDLLL SLC3A2 89 IFT81-001 ALASVIKEL IFT81 90 COG4-001 SLVAVELEKVCOG4 91 NCBP1-001 AMFENFVSV NCBP1

The present invention furthermore relates to the peptides according tothe present invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides consist or consist essentiallyof an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 65,and SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention.

The present invention further relates to the nucleic acid according tothe present invention that is DNA, cDNA, PNA, RNA or combinationsthereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine.

The present invention further relates to antibodies according to thepresent invention, and methods of making them.

The present invention further relates to T-cell receptors (TCR), inparticular soluble TCR (sTCRs), according to the present invention, andmethods of making them.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell.

The present invention further relates to the host cell according to thepresent invention wherein the antigen presenting cell is a dendriticcell.

The present invention further relates to a method of producing a peptideaccording to the present invention, the method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom the host cell or its culture medium.

The present invention further relates to an in vitro method forproducing activated cytotoxic T lymphocytes (CTL), the method comprisingcontacting in vitro CTL with antigen loaded human class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellfor a period of time sufficient to activate said CTL in an antigenspecific manner, wherein said antigen is any peptide according to thepresent invention.

The present invention further relates to the method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing said peptide containing SEQ IDNo. 1 to SEQ ID No. 92, preferably containing SEQ ID No. 1 to SEQ ID No.65 and SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92, or saidvariant amino acid sequence.

The present invention further relates to activated cytotoxic Tlymphocytes (CTL), produced by the method according to the presentinvention, which selectively recognize a cell which aberrantly expressesa polypeptide comprising an amino acid sequence according to the presentinvention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof cytotoxic T lymphocytes (CTL) as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament.

The present invention further relates to a use according to the presentinvention, wherein said medicament is a vaccine.

The present invention further relates to a use according to the presentinvention, wherein the medicament is active against cancer.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are lung cancer cells, gastric,gastrointestinal, colorectal, pancreatic or renal cancer cells, andglioblastoma cells.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention thatcan be used in the diagnosis and/or prognosis of lung, gastric,gastrointestinal, colorectal, pancreatic or renal cancer, andglioblastoma.

Further, the present invention relates to the use of these novel targetsfor cancer treatment.

Stimulation of an immune response is dependent upon the presence ofantigens recognised as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognising and destroying tumor cells. The isolation ofcytotoxic T-cells (CTL) from tumor-infiltrating cell populations or fromperipheral blood suggests that such cells play an important role innatural immune defences against cancer. CD8-positive T-cells inparticular, which recognise Class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 10amino acid residues derived from proteins or defect ribosomal products(DRIPS) located in the cytosol, play an important role in this response.The MHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

There are two classes of MHC-molecules: MHC class I molecules that canbe found on most cells having a nucleus. MHC molecules are composed ofan alpha heavy chain and beta-2-microglobulin (MHC class I receptors) oran alpha and a beta chain (MHC class II receptors), respectively. Theirthree-dimensional conformation results in a binding groove, which isused for non-covalent interaction with peptides. MHC class I presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, DRIPs and larger peptides. MHC class II moleculescan be found predominantly on professional antigen presenting cells(APCs), and primarily present peptides of exogenous or transmembraneproteins that are taken up by APCs during the course of endocytosis, andare subsequently processed. Complexes of peptide and MHC class Imolecules are recognized by CD8-positive cytotoxic T-lymphocytes bearingthe appropriate TCR (T-cell receptor), whereas complexes of peptide andMHC class II molecules are recognized by CD4-positive-helper-T cellsbearing the appropriate TCR. It is well known that the TCR, the peptideand the MHC are thereby present in a stoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Kobayashi et al., 2002; Qin et al., 2003; Gnjatic et al., 2003). At thetumor site, T helper cells, support a CTL friendly cytokine milieu(Mortara et al., 2006) and attract effector cells, e.g. CTLs, NK cells,macrophages, granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave surprisingly been found to express MHC class II molecules (Dengjelet al., 2006).

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CTL effector cells (i.e., CD8-positive T lymphocytes),CD4-positive T cells are sufficient for inhibiting manifestation oftumors via inhibition of angiogenesis by secretion of interferon-gamma(IFNγ).

Additionally, it was shown that CD4-positive T cells recognizingpeptides from tumor-associated antigens presented by HLA class IImolecules can counteract tumor progression via the induction of antibody(Ab) responses.

In contrast to tumor-associated peptides binding to HLA class Imolecules, only a small number of class II ligands of tumor associatedantigens (TAA) have been described to date.

Since the constitutive expression of HLA class II molecules is usuallylimited to cells of the immune system, the possibility of isolatingclass II peptides directly from primary tumors was not consideredpossible. However, Dengjel et al. were successful in identifying anumber of MHC Class II epitopes directly from tumors (WO 2007/028574, EP1 760 088 Bi; (Dengjel et al., 2006).

The antigens that are recognized by the tumor specific cytotoxic Tlymphocytes, that is, their epitopes, can be molecules derived from allprotein classes, such as enzymes, receptors, transcription factors, etc.which are expressed and, as compared to unaltered cells of the sameorigin, up-regulated in cells of the respective tumor.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+CTLs (ligand: MHC class I molecule+peptide epitope) or by CD4-positiveT-helper cells (ligand: MHC class II molecule+peptide epitope) isimportant in the development of tumor vaccines.

The present invention also relates to two new and very useful MHC classII peptides (according to SEQ ID NOs 76 and 77). These peptides areparticularly useful in the diagnosis and/or treatment of gastric cancer,NSCLC and other cancers over-expressing and/or over-presenting MMP12 andPOSTN respectively.

The present invention also relates to so-called length variants of theinventive MHC class II peptides according to SEQ ID NO 76 or 77. Asmentioned above, the peptide according to SEQ ID NO 76 consists of theamino acid sequence INNYTPDMNREDVDYAIR (MMP12-peptide), and the peptideaccording to SEQ ID NO 77 consists of the amino acid sequenceTNGVIHVVDKLLYPADT (POSTN-002-peptide). The length variants are generallyN- and/or C-terminally extended (between 1 and 5, preferably 1 to 10amino acids) or N- and/or C-terminally shortened (between 1 and 5 aminoacids) peptides, which still can bind to MHC, and elicit a cellularimmune response as described herein. As is known in the state of theart, peptides binding to class II proteins are not constrained in sizeand can vary from 11 to 30 amino acids in length. The peptide bindinggroove in the MHC class II molecules is open at both ends, which enablesbinding of peptides with relatively longer length. Though the “core”nine residues long segment contributes the most to the recognition ofthe peptide, the flanking regions are also important for the specificityof the peptide to the class II allele (see, for example, Meydan C, etal., Prediction of peptides binding to MHC class I and II alleles bytemporal motif mining. BMC Bioinformatics. 2013; 14 Suppl 2: S13. Epub2013 Jan. 21). Using the many software tools as available (e.g. asdescribed above), the person of skill in the art will be able toidentify the binding motif, and thus identify the possibilities forextensions and/or deletions of the MHC class II peptides according toSEQ ID NO 76 or 77, in order to create length variants.

For a peptide to trigger (elicit) a cellular immune response, it mustbind to an MHC-molecule. This process is dependent on the allele of theMHC-molecule and specific polymorphisms of the amino acid sequence ofthe peptide. MHC-class-I-binding peptides are usually 8-12 amino acidresidues in length and usually contain two conserved residues(“anchors”) in their sequence that interact with the correspondingbinding groove of the MHC-molecule. In this way each MHC allele has a“binding motif” determining which peptides can bind specifically to thebinding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules being expressed bytumor cells, they also have to be recognized by T cells bearing specificT cell receptors (TCR).

The antigens that are recognized by the tumor specific cytotoxic Tlymphocytes, that is, their epitopes, can be molecules derived from allprotein classes, such as enzymes, receptors, transcription factors, etc.which are expressed and, as compared to unaltered cells of the sameorigin, up-regulated in cells of the respective tumor.

The current classification of tumor associated antigens comprises thefollowing major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members or NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose; most are found inmelanomas and normal melanocytes. Many of these melanocytelineage-related proteins are involved in the biosynthesis of melanin andare therefore not tumor specific but nevertheless are widely used forcancer immunotherapy. Examples include, but are not limited to,tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.c) Overexpressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir overexpression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, Survivin, Telomerase or WT1.d) Tumor specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and to be used in a therapy,particular prerequisites must be fulfilled. The antigen should beexpressed mainly by tumor cells and not or in comparably small amountsby normal healthy tissues or in another preferred embodiment the peptideshould be over-presented by tumor cells as compared to normal healthytissues. It is furthermore desirable, that the respective antigen is notonly present in a type of tumor, but also in high concentrations (i.e.copy numbers of the respective peptide per cell). Tumor-specific andtumor-associated antigens are often derived from proteins directlyinvolved in transformation of a normal cell to a tumor cell due to afunction e.g. in cell cycle control or suppression of apoptosis.Additionally, downstream targets of the proteins directly causative fora transformation may be upregulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). In bothcases it is essential that epitopes are present in the amino acidsequence of the antigen, since such a peptide (“immunogenic peptide”)that is derived from a tumor associated antigen should lead to an invitro or in vivo T-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell with a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a tumorvaccine. The methods for identifying and characterizing the TAAs arebased on the use of CTL that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumors and normal tissues.

However, the identification of genes over-expressed in tumor tissues orhuman tumor cell lines, or selectively expressed in such tissues or celllines, does not provide precise information as to the use of theantigens being transcribed from these genes in an immune therapy. Thisis because only an individual subpopulation of epitopes of theseantigens are suitable for such an application since a T cell with acorresponding TCR has to be present and immunological tolerance for thisparticular epitope needs to be absent or minimal. In a very preferredembodiment of the invention it is therefore important to select onlythose over- or selectively presented peptides against which a functionaland/or a proliferating T cell can be found. Such a functional T cell isdefined as a T cell, which upon stimulation with a specific antigen canbe clonally expanded and is able to execute effector functions(“effector T cell”).

In case of TCRs and antibodies according to the invention theimmunogenicity of the underlying peptides is secondary. For TCRs andantibodies according to the invention the presentation is thedetermining factor.

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumor immunity. T-helper cell epitopes thattrigger a T-helper cell response of the T_(H1) type support effectorfunctions of CD8-positive killer T cells, which include cytotoxicfunctions directed against tumor cells displaying tumor-associatedpeptide/MHC complexes on their cell surfaces. In this waytumor-associated T-helper cell peptide epitopes, alone or in combinationwith other tumor-associated peptides, can serve as active pharmaceuticalingredients of vaccine compositions that stimulate anti-tumor immuneresponses.

Uses against further cancers are disclosed in the following descriptionof the proteins of the peptides according to the invention.

ATP-Binding Cassette, Sub-Family A (ABC1), Member 13 (ABCA13)

In human, the ATP-binding cassette (ABC) family of transmembranetransporters has at least 48 genes and 7 gene subfamilies. The predictedABCA13 protein consists of 5,058 amino acid residues making it thelargest ABC protein described to date (Prades et al., 2002). Knight etal. determined that ABCA13 protein is expressed in mouse and humanhippocampus and cortex, both regions relevant to schizophrenia andbipolar disorder (Knight et al., 2009). The ABCA13 gene maps tochromosome 7p12.3, a region that contains an inherited disorderaffecting the pancreas (Shwachman-Diamond syndrome) as well as a locusinvolved in T-cell tumor invasion and metastasis (INM7), and thereforeis a positional candidate for these pathologies (Prades et al., 2002).

Matrix Metallopeptidase 12 (Macrophage Elastase) (MMP12)

MMP12, also known as human metalloelastase (HME) or macrophagemetalloelastase (MME) is a zinc endopeptidase recognized for its abilityto degrade elastin. Apart from that, it has a broad substrate range,extending to other matrix proteins such as collagens, fibronectin,laminin, proteoglycans, and non-matrix proteins such asalpha-1-antitrypsin. In asthma, emphysema and chronic obstructivepulmonary disease (COPD), MMP12 may contribute to alveolar destructionand airway remodeling (Cataldo et al., 2003; Wallace et al., 2008).MMP12 has been implicated in macrophage migration, and as it cangenerate angiostatin from plasminogen, it contributes to inhibition ofangiogenesis (Chakraborti et al., 2003; Chandler et al., 1996; Sang,1998). Like other metalloproteinases, MMP12 is involved in physiologicalprocesses like embryogenesis, wound healing and the menstrual cycle(Chakraborti et al., 2003; Labied et al., 2009), but also inpathological processes of tissue destruction.

Although data are based on low numbers of patients in several cases,there is ample evidence in literature that MMP12 is frequentlyover-expressed in cancer (Denys et al., 2004; Hagemann et al., 2001; Maet al., 2009; Vazquez-Ortiz et al., 2005; Ye et al., 2008). However,data are controversial with respect to the impact of MMP12over-expression on clinical parameters and prognosis. While it may beinvolved in matrix dissolution and, thus, metastasis, it can alsoinhibit tumor growth through production of angiostatin, which negativelyimpacts angiogenesis (Gorrin-Rivas et al., 2000; Gorrin Rivas et al.,1998; Kim et al., 2004).

For lung cancer, consequences of MMP12 expression are controversial.MMP12 overexpression in epithelial cells has been reported ininflammation-triggered lung remodeling. MMP12 up-regulation may play arole in emphysema-to-lung cancer transition (Qu et al., 2009). Animalstudies suggest that MMP12 expression by stroma or macrophagessuppresses growth of lung tumors (Acuff et al., 2006; Houghton et al.,2006). However, there are also reports that MMP12 over-expression inlung tumors correlates with recurrence, metastatic disease and shorterrelapse-free survival after resection (Cho et al., 2004; Hofmann et al.,2005).

Dystonin (DST)

DST (BPAG1-e) encodes a member of the plakin protein family of adhesionjunction plaque proteins. BPAG1-e is expressed in epithelial tissue,anchoring keratin-containing intermediate filaments to hemidesmosomes(HDs). HDs are multiprotein adhesion complexes that promote epithelialstromal attachment in stratified and complex epithelia. Modulation oftheir function is of crucial importance in a variety of biologicalprocesses, such as differentiation and migration of keratinocytes duringwound healing and carcinoma invasion, in which cells become detachedfrom the substrate and acquire a motile phenotype (Litjens et al.,2006).

Malignant melanoma is one of the most aggressive types of tumor. BPAG1is expressed in human melanoma cell lines (A375 and G361) and normalhuman melanocytes. The levels of anti-BPAG1 auto-antibodies in the seraof melanoma patients were significantly higher than in the sera ofhealthy volunteers (p<0.01). Anti-BPAG1 auto-antibodies may be apromising marker for the diagnosis of melanoma (Shimbo et al., 2010).DST was associated with breast cancer invasion (Schuetz et al., 2006).The BPAG1 gene is likely to be involved in the proliferation, apoptosis,invasion and metastasis of nasopharyngeal carcinoma NPC (Fang et al.,2005).

Matrix-Remodeling Associated 5 (MXRA5)

MXRA5, also known as adlican, encodes an adhesion proteoglycan andbelongs to a group of genes involved in ECM remodeling and cell-celladhesion (Rodningen et al., 2008). Although the function of MXRA5 incancer is unknown, somatic mutations in MXRA5 have been identified intumors obtained from a variety of tissues such as skin, brain, lung, andovary. RT-PCR was performed on adlican (MXRA5) confirmed microarrayfindings of overexpression in colon cancers compared to normal colontissue (13 colorectal tumors and 13 normal tissues) (Zou et al., 2002).In a recent study, matrix-remodeling associated 5 was the second mostfrequently mutated gene in NSCLC (first is TP53) (Xiong et al., 2012).

Cyclin-Dependent Kinase 4 (CDK4)/Cyclin-Dependent Kinase 6 (CDK6)

CDK4 is a member of the Ser/Thr protein kinase family. It is a catalyticsubunit of the protein kinase complex that is important for cell cycleG1 phase progression. The activity of this kinase is restricted to theG1- to S phase transition during the cell cycle and its expression isprimarily controlled at the transcriptional level (Xiao et al., 2007).CDK4 and CDK6 enzymes and their regulators, e.g., cyclins, play criticalroles in embryogenesis, homeostasis, and cancerogenesis (Graf et al.,2010).

In lung cancer tissues the expression level of CDK4 protein wassignificantly increased compared to normal tissues (P<0.001). Patientswith higher CDK4 expression had a markedly shorter overall survival timethan patients with low CDK4 expression. Multivariate analysis suggestedthe level of CDK4 expression was an independent prognostic indicator(P<0.001) for the survival of patients with lung cancer. Furthermore,suppressing CDK4 expression also significantly elevated the expressionof cell cycle regulator p21 (Wu et al., 2011a). In lung cells thatexpress an endogenous K-Ras oncogene, ablation of Cdk4, but not Cdk2 orCdk6, induces an immediate senescence response. No such response occursin lungs expressing a single Cdk4 allele or in other K-Ras-expressingtissues. Targeting Cdk4 alleles in advanced tumors detectable bycomputed tomography scanning also induces senescence and prevents tumorprogression (Puyol et al., 2010).

Heterogeneous Nuclear Ribonucleoprotein H1 (H) (HNRNPH1)/HeterogeneousNuclear Ribonucleoprotein H2 (H′) (HNRNPH2)

These genes belong to the subfamily of ubiquitously expressedheterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNAbinding proteins and they complex with heterogeneous nuclear RNA(hnRNA). These proteins are associated with pre-mRNAs in the nucleus andappear to influence pre-mRNA processing and other aspects of mRNAmetabolism and transport.

hnRNPH activity appears to be involved in the pathogenesis andprogression of malignant gliomas as the center of a splicing oncogenicswitch, which might reflect reactivation of stem cell patterns andmediates multiple key aspects of aggressive tumor behavior, includingevasion from apoptosis and invasiveness (Lefave et al., 2011). Smallinterfering RNA-mediated knockdown of hnRNP H or A-Raf resulted inMST2-dependent apoptosis. In contrast, enforced expression of eitherhnRNP H or A-Raf partially counteracted apoptosis induced by etoposide(Rauch et al., 2010). Up-regulation of hnRNP H/H′ is found in a fewtissues that normally express low cytoplasmic levels of hnRNP H/H′, forexample, adenocarcinoma of the pancreas, hepatocellular carcinoma andgastric carcinoma (Honore et al., 2004).

Tetratricopeptide Repeat, Ankyrin Repeat and Coiled-Coil Containing 2(TANC2)

TANC family comprises TANC1 and TANC2, which was identified in 2005 (Hanet al., 2010). TANC family proteins are involved in the regulation ofdendritic spines, spatial learning, and embryonic development, asTANC1-deficiency in mice reduces spine density in the hippocampus andimpaired spatial learning, whereas TANC2-deficiency causes embryoniclethality. In contrast, overexpression of TANC1 and TANC2 in culturedneurons enhances the density of dendritic spines and excitatorysynapses. TANC1 and 2 proteins are mainly expressed in the brain, inwhich a significant proportion of protein is located in small-vesiclemembranes (Han et al., 2010).

Ring Finger Protein 213 (RNF213)

RNF213 encodes a protein containing a C3HC4-type RING finger domain,which is a specialized type of Zn-finger that binds two atoms of zincand is thought to be involved in mediating protein-protein interactions.

A research group provided evidence suggesting, for the first time, theinvolvement of RNF213 in genetic susceptibility to moyamoya disease (Liuet al., 2011b). Another study has shown that the RNF213 gene was relatedto moyamoya disease susceptibility in the Han Chinese population (Wu etal., 2012).

Solute Carrier Family 34 (Sodium Phosphate), Member 2 (SLC34A2)

SLC34A2 is a pH-sensitive sodium-dependent phosphate transporter.Upregulation of SLC34A2 gene expression in well-differentiated tumorsmay reflect cell differentiation processes during ovarian cancerogenesisand could serve as potential marker for ovarian cancer diagnosis andprognosis (Shyian et al., 2011). RT-PCR confirmed increased expressionof SLC34A2 in papillary thyroid cancer (Kim et al., 2010b). There wasalso a significantly increased gene expression of SLC34A2 among breastcancer tissues compared with normal tissues (Chen et al., 2010a).

SET and MYND Domain Containing 3 (SMYD3)

It was previously reported that upregulation of SMYD3, a histone H3lysine-4-specific methyl-transferase, plays a key role in theproliferation of colorectal carcinoma (CRC) and hepatocellular carcinoma(HCC). In another study, they reveal that SMYD3 expression is alsoelevated in the great majority of breast cancer tissues. Similarly toCRC and HCC, silencing of SMYD3 by small interfering RNA to this generesulted in the inhibited growth of breast cancer cells, suggesting thatincreased SMYD3 expression is also essential for the proliferation ofbreast cancer cells (Hamamoto et al., 2006). Knockdown of SMYD3 by RNAinterference down-regulates c-Met expression and inhibits cellsmigration and invasion induced by HGF (Zou et al., 2009). SMYD3 playscrucial roles in HeLa cell proliferation and migration/invasion, and itmay be a useful therapeutic target in human cervical carcinomas (Wang etal., 2008b).

Aldo-Keto Reductase Family 1, Member C1 (AKR1C1)/Aldo-Keto ReductaseFamily 1, Member C2 (AKR1C2)

AKR1C1 and AKR1C2 differ in only seven amino-acid residues (Le et al.,2010). AKR1C1 and AKR1C2 regulate the activity of androgens, estrogens,and progesterone, and the occupancy and transactivation of thecorresponding receptors (Penning et al., 2000; Steckelbroeck et al.,2004). The AKR1C enzymes, except AKR1C4 which is liver specific, areexpressed in different normal and diseased tissues and have thus beenrelated to several diseases, such as lung, breast, prostate, endometrialcancer, myeloid leukemia, and others (Brozic et al., 2011; Byrns et al.,2011). Sensitivity to cisplatin appeared to be associated with AKR1Clevels in epithelial lung cancer cell lines (Chen et al., 2010b) and inNSCLC patients (Kuang et al., 2012; Stewart, 2010). Thus, overexpressionof AKR1C is an indicator of poor prognosis and chemo-resistance in humannon-small lung cancer (NSCLC) (Wang et al., 2007). Overexpression ofAKR1C2 is also associated with disease progression in prostatic cancer(Huang et al., 2010). Depletion of AKR1C2 expression with RNAi inhibitstumorigenesis in vivo and in vitro, which strongly suggests that AKR1C2siRNA might play a critical role in blocking hepatocarcinogenesis(Dong-Dong, 2007).

Reticulocalbin 1, EF-Hand Calcium Binding Domain (RCN1)/Reticulocalbin3, EF-Hand Calcium Binding Domain (RCN3)

Reticulocalbin 1 is a calcium-binding protein located in the lumen ofthe ER. Immunohistochemical examination demonstrated a broaddistribution of RCN in various organs of fetuses and adults,predominantly in the endocrine and exocrine organs. Overexpression ofRCN may play a role in tumorigenesis, tumor invasion, and drugresistance (Fukuda et al., 2007). Reticulocalbin 1 (RCN1) is a cellsurface-associated protein on both endothelial (EC) and prostate cancer(PCa) cell lines. RCN1 expression on the cell surface was upregulated bytumor necrosis factor alpha treatment of bone-marrow endothelial cells(Cooper et al., 2008). RCN1 is up-regulated in colorectal carcinoma(CRC) and was localized in cancer cells or in stromal cells near thecancer cells. It could be a novel candidate for CRC marker (Watanabe etal., 2008). RCN3 is a member of the CREC(Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-handCa2+-binding proteins localized to the secretory pathway (Tsuji et al.,2006). In oligodendrogliomas RCN3 is suggested as a potentiallyimportant candidate gene. Though little is known about the function ofRCN3 (Drucker et al., 2009).

Interleukin 8 (IL8)

IL8 is a chemokine of the CXC family that is one of the major mediatorsof the inflammatory response. This chemokine is secreted by several celltypes. It functions as a chemoattractant, and is also a potentangiogenic factor. The CXC (ELR+) chemokines like IL8 induceangiogenesis and may be important in cancers that have an angiogenicphenotype such as NSCLC (Arenberg et al., 1997). Recently it was foundthat tumor derived IL8 acted as an attractant for circulating tumorcells to return to the original tumor (breast cancer, colon cancer, andmelanoma tumors), leading to a more aggressive tumor phenotype (Kim etal., 2009). IL-8 levels are associated with lung cancer risk severalyears before diagnosis. Combination of IL-8 and CRP are more robustbiomarkers in predicting subsequent lung cancer (Pine et al., 2011).Activating mutations of KRAS or EGFR upregulate IL-8 expression inNSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderlypatients, NSCLCs with pleural involvement, and KRAS-mutatedadenocarcinomas; and IL-8 plays a role in cell growth and migration inoncogenic KRAS-driven NSCLC (Sunaga et al., 2012).

Pyrimidinergic Receptor P2Y, G-Protein Coupled, 6 (P2RY6)

P2RY6 belongs to the family of G-protein coupled receptors. This familyhas several receptor subtypes with different pharmacologicalselectivity, which overlaps in some cases, for various adenosine anduridine nucleotides. The P2Y6 subtype is expressed at particularly highlevels in the placenta, suggesting that P2Y6 plays an important role inplacental function. However, the cellular localization of P2Y6 withinthe placenta is unknown. P2Y6 may play an important role introphoblastic development, differentiation, and neoplasia (Somers etal., 1999). An important role for the pyrimidine-activated P2Y receptorin the inflammatory response of lung epithelia was indicated (Schafer etal., 2003).

HECT, UBA and WWE Domain Containing 1, E3 Ubiquitin Protein Ligase(HUWE1)

HUWE1 encodes a member of the HECT E3 ubiquitin ligase family. The HECTdomain lies in the C-terminus and contains the active-site cysteinewhich forms an intermediate ubiquitin-thioester bond.

ARF-BP1 (HUWE1) is a critical mediator of both the p53-independent andp53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 mayserve as a potential target for therapeutic intervention in tumorsregardless of p53 status (Chen et al., 2005a). Inactivation of ARF-BP1stabilized p53 and induced apoptosis (Chen et al., 2006). HUWE1 (HectH9)is over-expressed in multiple human tumors and is essential forproliferation of a subset of tumor cells (Adhikary et al., 2005; Zhanget al., 2011a). In breast cancer HUWE1 correlated significantly withrelevant prognostic factors, and with clinical outcome (Confalonieri etal., 2009).

Versican (VCAN)

VCAN is a member of the aggrecan/versican proteoglycan family. VCAN isknown to associate with a number of molecules in the extracellularmatrix including hyaluronan, tenascin, fibulin-1, fibronectin, CD44 andL-selectin, fibrillin, integrin, and link protein (Zheng et al., 2004).VCAN is expressed in a variety of tissues. It is highly expressed in theearly stages of tissue development, and its expression decreases aftertissue maturation. Its expression is also elevated during wound repairand tumor growth (Ghosh et al., 2010). Knockdown in human lungadenocarcinoma (A549) cells of VCAN by RNA interference significantlyinhibited tumor growth in vivo but not in vitro (Creighton et al.,2005). VCAN is a direct target of p53. High expression of VCAN has alsobeen found in the peritumoral stromal tissue of early stage prostatecancers, and of breast cancers, and it is associated with an aggressivetumor behavior (Yoon et al., 2002).

Drosha, Ribonuclease Type III (DROSHA)

Drosha is a Class 2 RNase III enzyme responsible for initiating theprocessing of microRNA (miRNA), or short RNA molecules naturallyexpressed by the cell that regulate a wide variety of other genes byinteracting with the RNA-induced silencing complex (RISC) to inducecleavage of complementary messenger RNA (mRNA) as part of the RNAipathway. A microRNA molecule is synthesized as a long RNA primarytranscript known as a pri-miRNA, which is cleaved by Drosha to produce acharacteristic stem-loop structure of about 70 base pairs long, known asa pre-miRNA (Lee et al., 2003). Drosha exists as part of a proteincomplex called the Microprocessor complex, which also contains thedouble-stranded RNA binding protein Pasha (also called DGCR8) (Denli etal., 2004), which is essential for Drosha activity and is capable ofbinding single-stranded fragments of the pri-miRNA that are required forproper processing (Han et al., 2006). Human Drosha was cloned in 2000,when it was identified as a nuclear dsRNA ribonuclease involved in theprocessing of ribosomal RNA precursors (Wu et al., 2000). Drosha was thefirst human RNase III enzyme identified and cloned. The other two humanenzymes that participate in the processing and activity of miRNA are theDicer and Argonaute proteins. Both Drosha and Pasha are localized to thecell nucleus, where processing of pri-miRNA to pre-miRNA occurs. Thislatter molecule is then further processed by the RNase Dicer into maturemiRNAs in the cell cytoplasm (Lee et al., 2003). Drosha and other miRNAprocessing enzymes may be important in cancer prognosis (Slack andWeidhaas, 2008).

Pleckstrin Homology Domain Containing, Family A (PhosphoinositideBinding Specific) Member 8 (PLEKHA8)

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2=PLEKHA8)encodes a cytoplasmic lipid transferase with a plekstrin homology domainthat has been implicated in vesicle maturation and transport fromtrans-Golgi to the plasma membrane (Cao et al., 2009). The introductionof ribozymes targeting the FAPP2 gene in colon carcinoma cells inducedtheir apoptosis in the presence of Fas agonistic antibody. Also, FAPP2siRNA transfected glioma and breast tumor cells showed significantincreases in apoptosis (Tritz et al., 2009). Later studies havehighlighted a role for FAPP2 as lipid transfer protein involved inglycosphingolipid metabolism at the Golgi complex (D'Angelo et al.,2012). Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a keyrole in glycosphingolipid (GSL) production using its C-terminal domainto transport newly synthesized glucosylceramide away from thecytosol-facing glucosylceramide synthase in the cis-Golgi for furtheranabolic processing (Kamlekar et al., 2013).

Acetyl-CoA Carboxylase Alpha (ACACA)

ACACA is a biotin-containing enzyme which catalyzes the carboxylation ofacetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acidsynthesis (Tong and Harwood, Jr., 2006). ACACA up-regulation has beenrecognized in multiple human cancers, promoting lipogenesis to meet theneed of cancer cells for rapid growth and proliferation. Therefore,ACACA might be effective as a potent target for cancer intervention, andthe inhibitors developed for the treatment of metabolic diseases wouldbe potential therapeutic agents for cancer therapy (Wang et al., 2010a).Two studies have shown that silencing of ACACA by RNA interferencecauses growth inhibition and induces cell death almost to the sameextent as observed after silencing of FASN gene expression (Brusselmanset al., 2005; Chajes et al., 2006). TOFA (5-tetradecyloxy-2-furoicacid), an allosteric inhibitor of ACACA, is cytotoxic to lung cancercells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15 and induceapoptosis (Wang et al., 2009a). Another highly potent inhibitor ofACACA, soraphen A, blocks lipogenesis and enhances fatty acid oxidationin prostate cancer cells. Cancer cells stop proliferating and ultimatelydie (Beckers et al., 2007). These findings suggest that apart frommalonyl-CoA accumulation, inhibition of lipogenesis per se may causecancer cell death and that ACACA may be a target for antineoplastictherapy after all (Brusselmans et al., 2005).

Integrin, Alpha 11 (ITGA11)

Integrins play crucial roles in diverse cellular and developmentalprocesses, including cell growth, differentiation, and survival, as wellas carcinogenesis, cancer cell invasion, and metastases. Integrinalpha11 (ITGA11/alpha11) is localized to stromal fibroblasts andcommonly overexpressed in non-small-cell lung carcinoma (NSCLC). Thealpha11 mRNA was overexpressed in both lung adenocarcinoma and squamouscell carcinoma (Wang et al., 2002). It has been reported that alpha11plays an important role in the ability of fibroblasts to promote thegrowth of NSCLC cells in vivo, and such activity is partially mediatedby its regulation of IGF2 expression (Zhu et al., 2007). For NSCLCpatients' clinicopathological characteristics, the overexpression ofhMTH1, SPD, HABP 2, ITGA11, COL11A1, and CK-19 was significantlycorrelated with the pathological stage (p<0.05). In addition, theoverexpression of hMTH1, SPD, ITGA11, and COL11A1 was correlated withlymph node metastasis and poor prognosis (Chong et al., 2006).

Collagen, Type XII, Alpha 1 (COL12A1)

The COL12A1 gene encodes the alpha chain of type XII collagen, a memberof the FACIT (fibril-associated collagens with interrupted triplehelices) collagen family. Type XII collagen is a homotrimer found inassociation with type I collagen, an association that is thought tomodify the interactions between collagen I fibrils and the surroundingmatrix (Oh et al., 1992). COL12A1 may be involved in basement membraneregulation providing specific molecular bridges between fibrils andother matrix components (Thierry et al., 2004). COL12A1 is expressed inheart, placenta, lung, skeletal muscle and pancreas (Dharmavaram et al.,1998), in a variety of connective tissues including articular andepiphyseal cartilage (Gregory et al., 2001; Walchli et al., 1994; Wattet al., 1992). COL12A1 was down-regulated in tumors with highmicrosatellite instability when compared to the stable group with low ornull microsatellite instability (Ortega et al., 2010).

Elastase, Neutrophil Expressed (ELANE)

Neutrophil elastase (or leukocyte elastase) also known as ELA2 (elastase2, neutrophil) is a serine proteinase in the same family as chymotrypsinand has broad substrate specificity. Secreted by neutrophils duringinflammation, it destroys bacteria and host tissue (Belaaouaj et al.,2000). Human neutrophil elastase (ELANE), a main actor in thedevelopment of chronic obstructive pulmonary diseases, has been recentlyinvolved in non-small cell lung cancer progression. It can act atseveral levels (i) intracellularly, clearing for instance the adaptormolecule insulin receptor substrate-1 (IRS-1) (ii) at the cell surface,hydrolyzing receptors as CD40 (iii) in the extracellular space,generating elastin fragments i.e. morphoelastokines which potentlystimulate cancer cell invasiveness and angiogenesis (Moroy et al.,2012). Neutrophil elastase directly induced tumor cell proliferation inboth human and mouse lung adenocarcinomas by gaining access to anendosomal compartment within tumor cells, where it degraded insulinreceptor substrate-1 (IRS-1) (Houghton et al., 2010).

Serpin Peptidase Inhibitor, Clade B (Ovalbumin), Member 3 (SERPINB3)

Squamous cellular carcinoma antigen (SCCA), also called SERPINB3, is amember of the high molecular weight family of serine protease inhibitors(serpins) (Suminami et al., 1991). High levels have been reported incancer of the head and neck tissue and other epithelial cancers (Torre,1998). SCCA has been reported to be overexpressed in tumoral compared toperitumoral tissue, suggesting a role as a potential marker forhistological detection of HCC (Pontisso et al., 2004). Serpin B3/B4,particularly Serpin B4, appears to play an important role in aberrantepithelial proliferation. Evaluation of Serpin B3/B4 could haveprognostic value in predicting disease progression, especially inpatients with increased susceptibility to lung cancer (Calabrese et al.,2012). On one hand, SCCA1 (SERPINB3) inhibits cell death induced bylysosomal injury while, on the other hand, it sensitizes cells to ERstress by activating caspase-8 independently of the death receptorapoptotic pathway (Ullman et al., 2011). Some findings indicate thatSERPINB3 plays an important role in the induction of epidermal barrierdisruption. SERPINB3 may be a critical determinant of barrier functionin the epidermis (Katagiri et al., 2010).

Kinesin Family Member 26B (KIF26B)

A kinesin is a protein belonging to a class of motor proteins found ineukaryotic cells. Kinesins move along microtubule filaments, and arepowered by the hydrolysis of ATP (thus kinesins are ATPases). Kif26b, akinesin family gene, is a downstream target of Sall1 (Nishinakamura etal., 2011). Kif26b is essential for kidney development because itregulates the adhesion of mesenchymal cells in contact with uretericbuds. Overexpression of Kif26b in vitro caused increased cell adhesionthrough interactions with non-muscle myosin (Terabayashi et al., 2012;Uchiyama et al., 2010).

Ankylosis, Progressive Homolog (Mouse) (ANKH)

ANKH (human homolog of progressive ankylosis) regulates the transport ofinorganic pyrophosphate (PPi) through the cell membrane (Wang et al.,2008a). Some data suggest that ANKH expression and function in vitro andin vivo are repressed in hypoxic environments and that the effect isregulated by HIF-1 (Zaka et al., 2009). Human ANKH gene is expressed invivo in a tissue-specific manner, with highest levels of mRNA expressionfound in brain, heart, and skeletal muscle (Guo et al., 2001). Mutationsin the ANKH gene have been associated with autosomal dominantcraniometaphyseal dysplasia (Kornak et al., 2010). ANKH wassignificantly up-regulated in cervical cancer cell lines withamplifications as compared to cell lines without amplifications (Klothet al., 2007). Genomic amplification of regions on chromosome arm 5p hasbeen observed frequently in small cell lung cancer (SCLC), implying thepresence of multiple oncogenes on this arm. Coe et al. described theidentification of microdeletions that have escaped detection byconventional screens and the identification TRIO and ANKH as novelputative oncogenes (Coe et al., 2005).

Nuclear RNA Export Factor 1 (NXF1) In human cells, the mRNA exportfactor NXF1 resides in the nucleoplasm and at nuclear pore complexes(Zhang et al., 2011 b). The transport of mRNA from the site oftranscription in the nucleus to the site of translation in the cytoplasmis an essential process in eukaryotic gene expression. In human cells,the mRNA export factor NXF1 (also known as TAP) escorts mRNA transcriptsout of the nucleus by simultaneously binding mRNA, mRNA adaptorproteins, and phenylalanine-glycine (FG) repeats of the nuclear porecomplex (Kelly and Corbett, 2009). NXF1 is unique among nucleartransport factors, as it is a multidomain protein that bears nostructural or mechanistic resemblance to the karyopherin proteins thattransport protein cargos, tRNAs, and microRNAs through the NPC. mRNAexport by NXF1 is a process that occurs independent of the GTPase Ran(Gruter et al., 1998). Nuclear export of mRNPs is mediated by transportfactors such as NXF1 that bind mRNPs and mediate their translocationthrough the central channel of nuclear pores (NPC) using transientinteractions with FG-nucleoporins (Wickramasinghe et al., 2010). mRNAscan be transported by either bulk export pathways involving NXF1/TAP ormore specialized pathways involving chromosome region maintenance 1(CRM1) (Siddiqui and Borden, 2012).

Regulator of G-Protein Signaling 4 (RGS4)

RGS4 acts as a GTPase accelerating protein to modulate μ- and δ-opioidreceptor (MOR and DOR, respectively) signaling. Opioid agonist-inducedreduction in RGS4 occurs via the ubiquitin-proteasome pathway and maycontribute to the maintenance of cell homeostasis in themorphine-dependent state (Wang and Traynor, 2011). RGS4 plays animportant role in regulating beta-cell function (Ruiz, I et al., 2010).Xie et al. suggested RGS4 as a novel suppressor of breast cancermigration and invasion, important steps of metastatic cascades (Xie etal., 2009). RGS4 was overexpressed in thyroid carcinoma. The effectivedown-regulation of its expression levels in thyroid cancer cellssignificantly attenuated viability of thyroid cancer cells, indicatingthe significant role of RGS4 in thyroid carcinogenesis (Nikolova et al.,2008). RGS4 was differentially expressed in a human pancreatic tumorcell line and found to be a possible marker gene for local tumorinvasion and liver metastases in pancreatic carcinoma (Niedergethmann etal., 2007). RGS4 overexpression delayed and altered lung epithelial celltubulation by selectively inhibiting G protein-mediated p38 MAPKactivation, and, consequently, by reducing epithelial cellproliferation, migration, and expression of vascular endothelial growthfactor (VEGF) (Albig and Schiemann, 2005).

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2)

GFPT2 is involved in neurite outgrowth, early neuronal cell development,neuropeptide signaling/synthesis and neuronal receptor (Tondreau et al.,2008). Genetic variants in GFPT2 are associated with type 2 diabetes anddiabetic nephropathy (Zhang et al., 2004). Furthermore, the associationof SNPs in GFPT2 suggests that the gene involved in modulation ofoxidative pathway could be major contributor to diabetic chronic renalinsufficiency (Prasad et al., 2010). DNA methylation of the GFPT2 genewas validated in primary acute lymphoblastic leukemia (ALL) samples.Patients with methylation of multiple CpG islands had a worse overallsurvival (Kuang et al., 2008). GFPT2 plays a role in glutaminemetabolism and was observed to be more highly expressed in mesenchymalcell lines. Glutamine metabolism may play an important role in tumorprogression and inhibitors of cellular metabolic pathways may be a formof epigenetic therapy (Simpson et al., 2012).

Cerebral Endothelial Cell Adhesion Molecule (CERCAM)

CERCAM is localized at the surface of endothelial cells (Starzyk et al.,2000) and mapped on chromosome 9q34.11, a candidate region on 9q,identified as linked to familial idiopathic scoliosis (Miller et al.,2012). The CEECAM1 gene is widely transcribed in the nervous system andin several secretory tissues such as salivary glands, pancreas, liverand placenta (Schegg et al., 2009). The CERCAM protein is structurallysimilar to the ColGalT enzymes GLT25D1 and GLT25D2. But although itsfunction is still not known, it seems to be is functionally differentfrom the related GLT25D1 protein, and the protein does not function as aglycosyltransferase like GLT25D1 and GLT25D2 proteins (Perrin-Tricaud etal., 2011).

UDP-N-Acetyl-Alpha-D-Galactosamine: PolypeptideN-Acetylgalactosaminyl-Transferase 2 (GalNAc-T2) (GALNT2)

GALNT2, catalyze the first step in mucin-type O-glycosylation ofpeptides in the Golgi apparatus. These enzymes transferN-acetylgalactosamine (GalNAc) from UDP-GalNAc to the hydroxyl group ofserine or threonine in target proteins (Peng et al., 2010). GALNT2 wasexpressed constitutively and at low levels in most or all humanadenocarcinoma cell lines from pancreas, colon, stomach, and breastexamined (Sutherlin et al., 1997). Studies have shown that O-glycans andGALNT genes play critical roles in a variety of biological functions andhuman disease development. Risk of epithelial ovarian cancer (Terry etal., 2010) and coronary artery disease (Willer et al., 2008) have beenassociated with single nucleotide polymorphisms of GALNT2. Aberrantglycosylation of cell surface glycoprotein due to specific alterationsof glycosyltransferase activity is usually associated with invasion andmetastasis of cancer. GALNT2 is involved in tumor migration and invasionin gastric carcinomas (Hua et al., 2012), in hepatocellular carcinoma(HCC) (Wu et al., 2011b) and in human malignant glioma (Liu et al.,2011a).

Heterogeneous Nuclear Ribonucleoprotein M (HNRNPM)

The HNRNPM gene belongs to the subfamily of ubiquitously expressedheterogeneous nuclear ribonucleoproteins (hnRNPs). HNRNPM is an abundantcomponent of human hnRNP complexes that can influence pre-mRNA splicingby regulating its own pre-mRNA splicing (Hase et al., 2006) or byaffecting the regulation of alternative splicing of fibroblast growthfactor receptor 2 (Hovhannisyan and Carstens, 2007). Proteomic analysesof in vitro purified spliceosomes detected HNRNPM in thepre-spliceosomal H-complex and throughout the spliceosome assembly(Rappsilber et al., 2002; Wahl et al., 2009). HNRNPM is involved in thespliceosome machinery through its interaction with the CDC5L/PLRG1spliceosomal subcomplex (Lleres et al., 2010). In human cancer cells,some results show that, cytoplasmic retention of IMP-3 and HNRNPM leadsto significant drop in proliferation. A nuclear IMP-3-HNRNPM complex isimportant for the efficient synthesis of CCND1, D3 and G1 and for theproliferation of human cancer cells (Rivera et al., 2013).

Basonuclin 1 (BNC1)

Basonuclin is a zinc-finger protein with a highly restricted tissuedistribution (Tseng, 1998). Thus far, basonuclin has been detectedmainly in the basal keratinocytes of stratified squamous epithelia(skin, oral epithelium, esophagus, vagina, and cornea) and in thegametogenic cells of the testis and ovary (Tseng and Green, 1994; Weinerand Green, 1998). There is now considerable evidence that basonuclin isa cell-type-specific transcription factor for rRNA genes (rDNA). Thezinc fingers of basonuclin interact with three evolutionarily conservedsites within the rDNA promoter (Iuchi and Green, 1999; Tseng et al.,1999). Epigenetic regulation by CpG methylation has an important role intumorigenesis as well as in the response to cancer therapy. BNC1 washypomethylated in radioresistant H1299 human non-small cell lung cancer(NSCLC) cell lines. Suppression of BNC1 mRNA expression in H1299 cellsalso reduced the resistance of these cells to ionizing radiation (Kim etal., 2010a). Aberrant DNA methylation of BNC1 was also detected inchronic lymphocytic leukemia (CLL) samples (Tong et al., 2010). In RenalCell Carcinoma (RCC), methylation of BNC1 was associated with a poorerprognosis independent of tumor size, stage or grade (Morris et al.,2010).

FK506 Binding Protein 10, 65 kDa (FKBP10)

FK506-binding protein 10 (FKBP10) belongs to the FKBP-typepeptidyl-prolyl cis/trans isomerase family. It is located in endoplasmicreticulum and acts as molecular chaperones (Ishikawa et al., 2008;Patterson et al., 2000). It is highly expressed in lung development andcan be reactivated in a coordinated manner with extracellular matrixproteins after lung injury (Patterson et al., 2005).

Frizzled Family Receptor 1 (FZD1), Frizzled Family Receptor 2 (FZD2),Frizzled Family Receptor 7 (FZD7)

The genes FZD2, FZD1 and FZD7 are all from the ‘frizzled’ gene family;members of this gene family encode 7-transmembrane domain proteins thatare receptors for Wnt signaling proteins.

The expression of the FZD2 gene appears to be developmentally regulated,with high levels of expression in fetal kidney and lung and in adultcolon and ovary (Sagara et al., 1998; Zhao et al., 1995).

The FZD1 protein contains a signal peptide, a cysteine-rich domain inthe N-terminal extracellular region, 7 transmembrane domains, and aC-terminal PDZ domain-binding motif. The FZD1 transcript is expressed invarious tissues, including lung as well as heart, kidney, pancreas,prostate, and ovary (Sagara et al., 1998). The expression of frizzled 1and 2 receptors was found to be up-regulated in breast cancer(Milovanovic et al., 2004).

The FZD7 protein contains an N-terminal signal sequence, 10 cysteineresidues typical of the cysteine-rich extracellular domain of Fz familymembers, 7 putative transmembrane domains, and an intracellularC-terminal tail with a PDZ domain-binding motif. FZD7 gene expressionmay downregulate APC function and enhance beta-catenin-mediated signalsin poorly differentiated human esophageal carcinomas (Sagara et al.,1998; Tanaka et al., 1998).

ATPase, Ca++ Transporting, Cardiac Muscle, Fast Twitch 1 (ATP2A1),ATPase, Ca++ Transporting, Cardiac Muscle, Fast Twitch 2 (ATP2A2)

Both genes (ATP2A1 and ATP2A2) encode SERCA Ca(2+)-ATPases. Sarcoplasmicreticulum (SR)1/ER calcium ATPases (SERCAs) are calcium pumps thatcouple ATP hydrolysis with calcium transport across the SR/ER membrane(MacLennan et al., 1997). SERCAs are encoded by three homologous genes:SERCA1 (ATP2A1), SERCA2 (ATP2A2), and SERCA3 (Wu et al., 1995). Someevidence has emerged to show that SERCA may also have a direct impact onthe processes of apoptosis, differentiation, and cell proliferation(Chami et al., 2000; Ma et al., 1999; Sakuntabhai et al., 1999).

Mutations in ATP2A1, encoding SERCA1, cause some autosomal recessiveforms of Brody disease, characterized by increasing impairment ofmuscular relaxation during exercise (Odermatt et al., 1996).

ATP2A2 is an ATPase associated with Darier's disease, a rare, autosomaldominant hereditary skin disorder characterized by abnormalkeratinization and acantholysis (Huo et al., 2010). Germline alterationsof ATP2A2 may predispose to lung and colon cancer and an impaired ATP2A2gene might be involved in carcinogenesis (Korosec et al., 2006). In aSmall Cell Lung Cancer (H1339) and an Adeno Carcinoma Lung Cancer (HCC)cell line the ER Ca2+-content was reduced compared to normal humanbronchial epithelial. The reduced Ca2+-content correlated with a reducedexpression of SERCA 2 pumping calcium into the ER (Bergner et al.,2009). ATP2A2 could be potential prognostic markers for colorectalcancer CRC patients. It was detected in circulating tumor cells (CTCs),and the postoperative relapse was significantly correlated with geneoverexpression (Huang et al., 2012).

Laminin, Gamma 2 (LAMC2)

Laminins, a family of extracellular matrix glycoproteins, are the majornoncollagenous constituent of basement membranes. They have beenimplicated in a wide variety of biological processes including celladhesion, differentiation, migration, signaling, neurite outgrowth andmetastasis. The LAMC2 gene encodes the laminin-5 γ2 chain, which is partof laminin-5, one of the major components of the basement membrane zone.LAMC2 was frequently up-regulation by promoter demethylation in gastriccancer (Kwon et al., 2011). LAMC2 was found to be overexpressed inangiotropic melanoma areas vs. avascular melanoma areas (Lugassy et al.,2009). LAMC2 is a biomarker of bladder cancer metastasis, and itsexpression level was associated with tumor grade (Smith et al., 2009b).LAMB3 and LAMC2 genes were co-expressed in 21 of 32 non-SCLC cell lines(66%) but only in one of 13 SCLC cell lines (8%). Coexpression of theLAMB3 and LAMC2 genes was also observed in all 4 cases of primarynon-SCLC cells examined but not in the corresponding non-cancerous lungcells (Manda et al., 2000).

Heat Shock 70 kDa Protein 2 (HSPA2), Heat Shock 70 kDa Protein 8 (HSPA8)

HSPA2 has been identified as a potential cancer-promoting proteinexpressed at abnormal levels in a subset of human cancers, such asbreast cancer (Mestiri et al., 2001), cervical cancer (Garg et al.,2010a), bladder urothelial cancer (Garg et al., 2010b), nasopharyngealcarcinoma (Jalbout et al., 2003) and malignant tumors (Chouchane et al.,1997). Some level of the HSPA2 gene activity was also observed in celllines derived from several human cancers (Scieglinska et al., 2008),while silencing of the HSPA2 gene in cancer cells led to growth arrestand decrease in tumorigenic potential (Rohde et al., 2005; Xia et al.,2008). Furthermore, polymorphism in the HSPA2 gene is associated with anincrease in the risk of developing lung cancer (Wang et al., 2010b).Overexpression of HSPA2 is correlated with increased cell proliferation,poor differentiation and lymph node metastases in human breast cancer,cervical cancer and bladder urothelial cancer (Garg et al., 2010a; Garget al., 2010b; Mestiri et al., 2001).

The HSPA8 gene encodes a member of the heat shock protein 70 familyHsc70, which contains both heat-inducible and constitutively expressedmembers. HSPA8 binds to nascent polypeptides to facilitate correctprotein folding (Beckmann et al., 1990). Hsc70 function as molecularchaperones, assisting in protein synthesis, folding, assembly,trafficking between cellular compartments, and degradation (Bukau andHorwich, 1998; Hard and Hayer-Hartl, 2002). Hsc70 is expressed innon-malignant mammary cells as well as breast cancer cells (Kao et al.,2003; Vargas-Roig et al., 1998) and the overexpression of Hsp/hsc70 inchemoresistant cancer cells (Ciocca et al., 1992; Lazaris et al., 1997)has prompted studies about possible clinical markers of these proteins(Ciocca and Calderwood, 2005). There is a potential role of thissecreted hsc70 chaperone in cell proliferation that might account forthe higher tumor growth of cancer cells overexpressing cathepsin D(Nirde et al., 2010). Furthermore Ruisin et al. reported an associationbetween a polymorphism of this gene and lung cancer risk (Rusin et al.,2004).

Vacuolar Protein Sorting 13 Homolog B (Yeast) (VPS13B)

VPS13B was identified as a peripheral membrane protein localized to theGolgi complex, where it overlaps with the cis-Golgi matrix proteinGM130. Consistent with its subcellular localization, VPS13B depletionusing RNAi causes fragmentation of the Golgi ribbon into ministacks(Seifert et al., 2011). Kolehmainen et al. (2003) identified the COH1gene, also known as VPS13B, within the Cohen syndrome critical region onchromosome 8q22 (Kolehmainen et al., 2003). Loss-of-function mutationsin the gene VPS13B lead to autosomal recessive Cohen syndrome (Seifertet al., 2011). Mutations of VPS13B and other genes were described ingastric and colorectal cancers with microsatellite instability (An etal., 2012).

CSE1 Chromosome Segregation 1-Like (Yeast) (CSE1L)

The cellular apoptosis susceptibility (CSE1L) gene has been demonstratedto regulate multiple cellular mechanisms including the mitotic spindlecheck point as well as proliferation and apoptosis. CSE1L is located inboth the cytoplasm and the nuclei of cells. Nuclear CSE1L regulates thetranscriptional activity of the p53 protein, a major tumor suppressorprotein (Rao et al., 2011; Tanaka et al., 2007). Cytoplasmic CSE1L isassociated with microtubules; this association has been shown tostimulate the extension of invadopodia and to enhance the migration oftumor cells (Tai et al., 2010). CSE1L is highly expressed in mostcancers, such as benign and malignant cutaneous melanocytic lesions(Boni et al., 1999), endometrial carcinoma (Peiro et al., 2001), ovariancarcinoma (Brustmann, 2004), breast cancer (Behrens et al., 2001),urinary bladder urothelial carcinomas (Chang et al., 2012), and itsexpression has been shown to correlate with cancer progression.Silencing of CSE1L may be a potential therapeutic approach for coloncancer (Zhu et al., 2013).

Dihydropyrimidinase-Like 4 (DPYSL4)

Dihydropyrimidinase-related protein 4 (DPYSL4) is a known regulator ofhippocampal neuron development. DPYSL4 is involved in growth regulation,polarization and differentiation of dental epithelial cells during toothgerm morphogenesis (Yasukawa et al., 2013). Some studies showed DPYSL4'srole in attenuating neurite outgrowth possibility through inhibitingmicrotubule polymerization, and also revealed its novel association withvimentin during nuclear condensation prior to neuronal death (Aylsworthet al., 2009). The p53 tumor suppressor gene, which is frequentlymutated in a wide variety of tumors, plays an important role inmaintaining genomic integrity. Both mRNA and protein expressions ofDPYSL4 were specifically induced by anticancer agents in p53-proficientcells. DPYSL4 is an apoptosis-inducible factor controlled by p53 inresponse to DNA damage (Kimura et al., 2011).

Sec61 Gamma Subunit (SEC61G)

SEC61γ, a heterotrimeric protein channel comprising the subunits SEC61α,β, and γ, is a member of the SEC61 translocon (Greenfield and High,1999). The SEC61 complex forms a transmembrane pore for thetranslocation of nascent polypeptides into the ER lumen, as well as theintegration of transmembrane proteins into the ER bilayer (Osborne etal., 2005). SEC61γ is required for tumor cell survival, and for thecellular response to endoplasmic reticulum stress. Furthermore it ishighly overexpressed in malignant cells and near absent in normal cells(Lu et al., 2009). Knocking down SEC61γ expression resulted in apoptosisand abrogation of EGFR/AKT survival signaling (Lu et al., 2009) as wellas to growth inhibition of the tumor cells (Neidert et al., 2012).

ORM1-Like 1 (S. cerevisiae) (ORMDL1)

The human genes (ORMDL1, ORMDL2 and ORMDL3) are expressed ubiquitouslyin adult and fetal tissues. They encode transmembrane proteins anchoredin the endoplasmic reticulum which are likely involved in proteinfolding in the ER By genomic sequence analysis, Hjelmqvist et al. (2002)mapped the ORMDL1 gene to chromosome 2q32.2 (Hjelmqvist et al., 2002).ORMDL proteins are the primary regulators of ceramide biosynthesis inmammalian cells (Siow and Wattenberg, 2012). ORMDL1 is specificallydown-regulated in association with presenilin 1 (PS1) mutations (Arakiet al., 2008).

Pecanex-Like 3 (Drosophila) (PCNXL3)

Pecanex-like protein 3 (PCNXL3) is a multi-pass membrane protein; itbelongs to the pecanex family.

The PCNXL3 gene was mapped to the chromosomal region 11q12.1-q13. Threenovel human tumor-associated translocation breakpoints were located inthe chromosome 11q13 region between the markers D11S4933 and D11S546.Thus PCNXL3 might be a 11q13-associated disease gene (van et al., 2000).

Small Nuclear Ribonucleoprotein 200 kDa (U5) (SNRNP200)

Pre-mRNA splicing is catalyzed by the spliceosome, a complex ofspecialized RNA and protein subunits that removes introns from atranscribed pre-mRNA segment. The spliceosome consists of small nuclearRNA proteins (snRNPs) U1, U2, U4, U5 and U6, together with approximately80 conserved proteins. SNRNP200 is a gene required for unwinding of theU4/U6 duplex, a step essential for catalytic activation of thespliceosome (Maeder et al., 2009). SNRNP200 expression was detected inheart, brain, placenta, lung, liver, skeletal muscle, kidney, andpancreas (Zhao et al., 2009). Mutations in SNRNP200 have recently beendiscovered to be associated with autosomal dominant retinitis pigmentosa(adRP) (Benaglio et al., 2011; Liu et al., 2012).

SAM Domain, SH3 Domain and Nuclear Localization Signals 1 (SAMSN1)

SAMSN1 is a member of a novel gene family of putative adaptors andscaffold proteins containing SH3 and SAM (sterile alpha motif) domains.SAMSN1 is expressed in hematopoietic tissues, muscle, heart, brain,lung, pancreas, endothelial cells and myelomas. Endogenous SAMSN1expression was shown to be up-regulated in primary B cells upondifferentiation and proliferation-inducing stimuli, and transductionexperiments suggest a stimulatory role for SAMSN1 in B celldifferentiation to plasma cells (Brandt et al., 2010). Cell lines andprimary cells from acute myeloid leukemia and multiple myeloma patientsexpress SAMSN1 (Claudio et al., 2001). SAMSN1 was down-regulated in thelarge cell lung carcinoma cell line Calu-6 (Yamada et al., 2008). SAMSN1was differentially expressed in ulcerative colitis-associated cancer(Watanabe et al., 2011).

Signal Transducer and Activator of Transcription 2, 113 kDa (STAT2)

STAT2 as a novel contributor to colorectal and skin carcinogenesis thatmay act to increase the gene expression and secretion ofpro-inflammatory mediators, which in turn activate the oncogenic STAT3signaling pathway (Gamero et al., 2010). STAT2 is a critical mediator inthe activation of type I IFN-induced apoptosis. More importantly,defects in the expression or nuclear localization of STAT2 could lessenthe efficacy of type I IFN immunotherapy (Romero-Weaver et al., 2010).Lower expression of STAT2 in low grade astrocytomas were detected whencomparing with high grade astrocytomas. The results showed existingrelationship between STAT and PPARgamma signaling in glial tumors andfurther support expected important role of STATs in regulation of growthand differentiation in these tumors (Ehrmann et al., 2008).

CCR4-NOT Transcription Complex, Subunit 1 (CNOT1)

The human CCR4-NOT deadenylase complex consists of at least nineenzymatic and non-enzymatic subunits. CNOT1 has an important role inexhibiting enzymatic activity of the CCR4-NOT complex, and thus iscritical in control of mRNA deadenylation and mRNA decay. CNOT1depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in theincrement of translation causing ER stress-mediated apoptosis. Ito etal. conclude that CNOT1 contributes to cell viability by securing theactivity of the CCR4-NOT deadenylase (Ito et al., 2011). siRNA-mediateddepletion of endogenous CNOT1 or other Ccr4-Not subunits in breastcancer cells results in deregulation of ERalpha target genes (increasedinduction of ERα target genes TTF1 and c-Myc). These findings define afunction for the human Ccr4-Not complex as a transcriptional repressorof nuclear receptor signaling that is relevant for the understanding ofmolecular pathways involved in cancer (Winkler et al., 2006).

Serine Hydroxymethyltransferase 2 (Mitochondrial) (SHMT2)

The SHMT2 gene encodes the mitochondrial form of a pyridoxalphosphate-dependent enzyme that catalyzes the reversible reaction ofserine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. The encoded product is primarily responsible forglycine synthesis. In a polygenic disease such as lung cancer, gene-geneinteractions are expected to play an important role in determining thephenotypic variability of the diseases. Interactions between MTHFR677,MTHFR1298, and SHMT polymorphisms may have a significant impact ongenetic instability in lung cancer patients. It was shown that withregard to cytogenetic alterations lymphocytes from lung cancer patientsexposed to the tobacco-specific carcinogen4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone [NNK] had considerablyincreased frequency of cytogenetic damage in presence of MTHFR 677,MTHFR 1298, and SHMT allelic variants (Piskac-Collier et al., 2011).Pharmacogenomic studies on the role of polymorphism of the SHMT gene inthe efficacy of 5-FU and FOLFIRI protocols of colorectal cancer patientsrevealed a significant effect resulting in altered overall survival aswell (Timar et al., 2006).

Jun B Proto-Oncogene (JUNB)

JunB is a member of the AP-1 (activator protein-1) family of dimerictranscription factors. The transcription factor AP-1 is involved incellular proliferation, transformation and death (Shaulian and Karin,2002). JunB might be regulated through an NF-êB pathway andup-regulation of JunB induced by HGF might play an important role in theregulation of cell proliferation and cell invasion through MMP-9expression (Lee and Kim, 2012). JunB seems to play an oncogenic role inlymphomas, particularly in Hodgkin's lymphomas (Shaulian, 2010). JunB isan essential upstream regulator of p16 and contributes to maintain cellsenescence that blocks malignant transformation of TAC. JunB thusapparently plays an important role in controlling prostatecarcinogenesis (Konishi et al., 2008). JunB promotes tumor invasivenessand enhances angiogenesis in VHL-defective ccRCCs (Kanno et al., 2012).

Transforming, Acidic Coiled-Coil Containing Protein 3 (TACC3)

TACC3 exists in a complex with ch-TOG (colonic and hepatic tumorover-expressed gene) and clathrin that cross-links microtubules inkinetochore fibres. TACC3 is expressed in certain proliferative tissuesincluding testis, lung, spleen, bone marrow, thymus and peripheral bloodleukocytes. TACC3 expression is altered in some human tumor types. Incells, TACC3 is localized to both centrosomes and spindle microtubulesbut not at astral microtubules (Hood and Royle, 2011). TACC3 expressionwas correlated with p53 expression, and patient whose tumors highlyexpressed TACC3 and p53 had a significantly poorer prognosis thanpatients whose tumors had low-level expression for both immunostainings(P=0.006). It is suggested that increase in TACC3 may impart aproliferative advantage to NSCLC and contribute to tumor progression,and that TACC3 expression is a strong prognostic indicator of clinicaloutcome in NSCLC (Jung et al., 2006). Tacc3 may be a negative regulatorof the Notch signalling pathway (Bargo et al., 2010).

RAD54 Homolog B (S. cerevisiae) (RAD54B)

DNA repair and recombination protein RAD54B is a protein that in humansis encoded by the RAD54B gene. RAD54 binds to double-stranded DNA, anddisplays ATPase activity in the presence of DNA. The human RAD54Bprotein is a paralog of the RAD54 protein, which plays important rolesin homologous recombination. Homologous recombination (HR) is essentialfor the accurate repair of DNA double-strand breaks (DSBs) (Sarai etal., 2008). Knockdown of RAD54B, a gene known to be somatically mutatedin cancer, causes chromosome instability (CIN) in mammalian cells(McManus et al., 2009). RAD54B elevated gene expression is significantlyassociated with shorter time-to-progression and poor OS in GBM patients(Grunda et al., 2010).

Eukaryotic Translation Elongation Factor 2 (EEF2)

EEF2 encodes a member of the GTP-binding translation elongation factorfamily. This protein is an essential factor for protein synthesis. Itpromotes the GTP-dependent translocation of the nascent protein chainfrom the A-site to the P-site of the ribosome. EEF2 was highly expressedin lung adenocarcinoma (LADC), but not in the neighboring non-tumor lungtissue. It is suggested that eEF2 is an anti-apoptotic marker in LADC,because patients with high eEF2 expression had a significantly higherincidence of early tumor recurrence, and a significantly worseprognosis. Silencing of eEF2 expression increased mitochondrialelongation, cellular autophagy and cisplatin sensitivity. Moreover, eEF2was SUMOylated in LADC cells, and eEF2 SUMOylation correlated with drugresistance (Chen et al., 2011a). EEF2 is an attractive target for cancertherapy because inhibiting EEF2 causes the rapid arrest of proteinsynthesis, inducing apoptosis and leading ultimately to cell death.siRNA-induced silencing of EEF2 resulted in specific cytotoxicity oftumor cells (Chen et al., 201 b; Wullner et al., 2008).

Cydin A2 (CCNA2)

CCNA2 belongs to the highly conserved cyclin family. Cyclins function asregulators of CDK kinases. Different cyclins exhibit distinct expressionand degradation patterns which contribute to the temporal coordinationof each mitotic event (Deshpande et al., 2005). Human cyclin A2 is a keyregulator of S phase progression and entry into mitosis. CCNA2 binds andactivates CDC2 or CDK2 kinases, and thus promotes both cell cycle G1/Sand G2/M transitions (Honda et al., 2012). Mutations, amplifications andoverexpression of this gene, which alters cell cycle progression, areobserved frequently in a variety of tumors and may contribute totumorigenesis (Cooper et al., 2009; Kars et al., 2011; Kim et al., 2011;Tompkins et al., 2011). Furthermore, it is described that CCNA2expression is associated with a poor prognosis in several types ofcancer (Yasmeen et al., 2003) and that elevated expression of cyclin Acorrelated to shorter survival periods (Dobashi et al., 1998).

Neuroepithelial Cell Transforming 1 (NET1) 41

NET1 is part of the family of Rho guanine nucleotide exchange factors.Members of this family activate Rho proteins by catalyzing the exchangeof GDP for GTP. The protein encoded by NET1 interacts with RhoA withinthe cell nucleus and may play a role in repairing DNA damage afterionizing radiation.

The NET1 gene, but not opioid receptors, is expressed in breastadenocarcinoma cells and may facilitate their migration (Ecimovic etal., 2011). NET1 is up-regulated in gastric cancer (GC) tissue anddrives the invasive phenotype of this disease (Srougi and Burridge,2011). NET1 plays an important role in GC cell migration and invasion,key aspects of GC progression (Bennett et al., 2011). The higherexpressions of RhoC and NET1 in human prostate cancers after short-termendocrine therapy suggest that RhoC and NET1 may become therapeutictargets during endocrine therapy (Kawata et al., 2012).

Chromosome 11 Open Reading Frame 24 (C11orf24)

C11orf24 was identified by Twells et al (2001). The C11orf24 gene has noknown similarity to other genes, and its function is unknown. Northernblot analysis detected high expression of a 1.9-kb transcript in heart,placenta, liver, pancreas, and colon. Lower levels were detected inbrain, lung, skeletal muscle, kidney, spleen, prostate, testis, ovary,and small intestine, and very low levels were detected in thymus andleukocytes (Twells et al., 2001). The 449 amino acid long proteinC11orf24 is located on the chromosomal region 11q13. This region isdescribed as a multi-cancer susceptibility region (Gudmundsson et al.,2009; Purdue et al., 2011).

Regulator of Chromosome Condensation 1 (RCC1)

Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotideexchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1on chromatin is critical for nucleocytoplasmic transport, mitoticspindle assembly and nuclear envelope formation (Hitakomate et al.,2010). Some data suggested that chromosomal binding of the mitoticregulators such as RCC1, Mad2 and survivin is essential for mitoticprogression (Ho et al., 2008). Wong et al. have found that the nuclearRanGTP level is diminished during the early stages of apoptosis, whichcorrelates with immobilization of RCC1 on the chromosomes. Therefore,they propose that RCC1 reads the histone code created bycaspase-activated Mst1 to initiate apoptosis by reducing the level ofRanGTP in the nucleus (Wong et al., 2009).

Melanoma Antigen Family F, 1 (MAGEF1)

Most known members of the MAGE (melanoma-associated antigen) superfamilyare expressed in tumors, testis and fetal tissues, which has beendescribed as a cancer/testis expression, pattern (MAGE subgroup I).Peptides of MAGE subgroup I have been successfully used in peptide andDC vaccination (Nestle et al., 1998; Marchand et al., 1999; Marchand etal., 1999; Marchand et al., 1995; Thumer et al., 1999). In contrast,some MAGE genes (MAGE subgroup II), such as MAGEF1, are expressedubiquitously in all adult and fetal tissues tested and also in manytumor types including ovarian, breast, cervical, melanoma and leukemia(Nestle et al., 1998; Marchand et al., 1999; Marchand et al., 1999;Marchand et al., 1995; Thurner et al., 1999). Nevertheless,overexpression of MAGEF1 could be detected in NSCLC (Tsai et al., 2007)and in 79% of a cohort of Taiwanese colorectal cancer patients (Chung etal., 2010).

Non-SMC Condensin I Complex, Subunit D2 (NCAPD2)

Condensins are heteropentameric complexes that were first identified asstructural components of mitotic chromosomes. NCAPD2 is an essentialcomponent of the human condensin complex required for mitotic chromosomecondensation. NCAPD2 depletion affects chromosome alignment in metaphaseand delays entry into anaphase (Watrin and Legagneux, 2005). Recentlinkage and association studies have implicated the chromosome 12p13locus as possibly harboring genetic variants predisposed to Alzheimer'sdisease (AD). Single marker association revealed the two SNPs in NCAPD2(rs7311174 and rs2072374) as showing nominal significant p values(p=0.0491 and 0.0116, respectively). These genetic analyses provideevidence that the chromosome 12p13 locus is associated with AD inChinese (Li et al., 2009).

Chromosome 12 Open Reading Frame 44 (C12orf44)

By searching databases for orthologs of a Drosophila Atg13-interactingprotein, Mercer et al. (2009) identified human ATG101, also known asC12orf44 (Mercer et al., 2009). The ATG101 gene was mapped to chromosome12q13.13. The deduced 218 amino acid protein was predicted to be acytosolic hydrophilic protein (Hosokawa et al., 2009). Macroautophagy isa catabolic process for lysosome-mediated degradation of cytoplasmicproteins, organelles, and macromolecules. ATG proteins, such as ATG101,are required for formation of autophagosomes, double-membrane vesiclesthat surround and sequester cytoplasmic cargo prior to fusion withlysosomes. ATG101 (C12orf44) is essential for autophagy (Mercer et al.,2009).

HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 4 (HERC4)

HERC4 belongs to the HERC family of ubiquitin ligases, all of whichcontain a HECT domain and at least 1 RCC1 (MIM 179710)-like domain(RLD). The 350-amino acid HECT domain is predicted to catalyze theformation of a thioester with ubiquitin before transferring it to asubstrate, and the RLD is predicted to act as a guanine nucleotideexchange factor for small G proteins (Hochrainer et al., 2005). E3ubiquitin ligase Herc4, though ubiquitously expressed in all tissues, ismost highly expressed in the testis, specifically during spermiogenesis.Herc4 ligase is required for proper maturation and removal of thecytoplasmic droplet for the spermatozoon to become fully functional(Rodriguez and Stewart, 2007).

Insulin-Like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3)

IGF2BP3 is a member of the insulin-like growth factor-II mRNA-bindingprotein family, implicated in mRNA localization, turnover andtranslational control. The protein contains several KH (K-homologous)domains, which are important in RNA binding and are known to be involvedin RNA synthesis and metabolism. Expression occurs mainly duringembryonic development and has been described for some tumors. Thus,IGF2BP3 is considered to be an oncofoetal protein (Liao et al., 2005).IGF2BP3 may promote tumor cell proliferation by enhancing IGF-II proteinsynthesis and by inducing cell adhesion and invasion throughstabilization of CD44 mRNA (Findeis-Hosey and Xu, 2012). Moreover,IGF2BP3 expression has been studied in many human neoplasms with growingevidence that it mediates migration, invasion, cell survival and tumormetastasis (Jeng et al., 2009; Kabbarah et al., 2010; Li et al., 2011;Liao et al., 2011; Lu et al., 2011; Hwang et al., 2012; Samanta et al.,2012) and it might also be implicated in angiogenesis (Suvasini et al.,2011; Chen et al., 2012). In lung adenocarcinomas, a higher frequency ofIGF2BP3 expression can be detected in moderately or poorlydifferentiated adenocarcinomas, which may be associated with anaggressive biological behavior (Findeis-Hosey et al., 2010; Beljan etal., 2012; Findeis-Hosey and Xu, 2012).

Cell Division Cycle 6 Homolog (S. cerevisiae) (CDC6)

CDC6 protein functions as a regulator at the early steps of DNAreplication. It localizes in cell nucleus during cell cycle G1, buttranslocates to the cytoplasm at the start of S phase. Further, CDC6 issupposed to regulate replication-checkpoint activation through theinteraction with ATR in higher eukaryotic cells (Yoshida et al., 2010).CDC6 is essential for DNA replication and its deregulation is involvedin carcinogenesis. It was found that CDC6 down-regulation by RNAinterference (RNAi) prevented cell proliferation and promoted apoptosis(Lau et al., 2006). Overexpression of CDC6 was found in several cancers.Among the cancer types over-expressing CDC6 are gastric cancer(Tsukamoto et al., 2008), brain tumors (Ohta et al., 2001), oralsquamous cell carcinoma (Feng et al., 2008), cervical carcinoma (Wang etal., 2009b) and malignant mesothelioma (Romagnoli et al., 2009).

Fibroblast Activation Protein, Alpha (FAP)

Fibroblast activation protein (FAP) is a type II integral membraneglycoprotein belonging to the serine protease family. The putativeserine protease activity of FAP alpha and its in vivo induction patternmay indicate a role for this molecule in the control of fibroblastgrowth or epithelial-mesenchymal interactions during development, tissuerepair, and epithelial carcinogenesis (Scanlan et al., 1994). Mostnormal adult tissues and benign epithelial tumors show little or nodetectable FAP expression. However, FAP expression is detected in thestroma of over 90% of malignant breast, colorectal, lung, skin andpancreatic tumors, fibroblasts of healing wounds, soft tissue sarcomas,and some fetal mesenchymal cells. FAP has a potential role in cancergrowth and metastasis through cell adhesion and migration processes, aswell as rapid degradation of ECM components. Thus, it is present ontumor cells invading the ECM, and endothelial cells involved inangiogenesis, but is not expressed in inactive cells of the same type(Dolznig et al., 2005; Kennedy et al., 2009; Rettig et al., 1993; Rettiget al., 1994; Scanlan et al., 1994; Zhang et al., 2010a).

Wingless-Type MMTV Integration Site Family, Member 5A (WNT5A)

In general, Wnt5a regulates a variety of cellular functions, such asproliferation, differentiation, migration, adhesion and polarity(Kikuchi et al., 2012). It is expressed in undifferentiated humanembryonic stem cells (Katoh, 2008). WNT5A is classified as anon-transforming WNT family member whose role in carcinogenesis is stillambiguous. It exhibits tumor suppressor activities in some cancers(thyroid, brain, breast and colorectum), but is aberrantly up-regulatedin cancers of lung, stomach and prostate (Li et al., 2010). OncogenicWNT5A activates canonical WNT signaling in cancer stem cells forself-renewal, and non-canonical WNT signaling at the tumor-stromalinterface for invasion and metastasis (Katoh and Katoh, 2007).Expression of WNT5A has been described for a variety of tumor entities.For example, abnormal protein expression of Wnt5a was observed in 28% ofprostate cancer where it promotes aggressiveness (Yamamoto et al.,2010). Furthermore, WNT5A over-expression is described to be associatedwith poor prognosis and/or increasing tumor grade in ovarian cancer(Badiglian et al., 2009), melanoma (Da Fomo et al., 2008; Weeraratna etal., 2002), GBM (Yu et al., 2007), lung cancer (Huang et al., 2005) andpancreatic cancer (Ripka et al., 2007). In HCC, it seems that thecanonical Wnt signaling pathway contributes to tumor initiation and thenoncanonical signaling to tumor progression (Yuzugullu et al., 2009).

TPX2, Microtubule-Associated, Homolog (Xenopus laevis) (TPX2)

TPX2 is a spindle assembly factor. It is required for normal assembly ofmitotic spindles and of microtubules during apoptosis. TPX2 is requiredfor chromatin and/or kinetochore dependent microtubule nucleation (Birdand Hyman, 2008; Moss et al., 2009). Newly synthesized TPX2 is requiredfor nearly all Aurora A activation and for full p53 synthesis andphosphorylation in vivo during oocyte maturation (Pascreau et al.,2009). TPX2 is a cell cycle-associated protein which is overexpressed inmany tumor types, such as meningiomas (Stuart et al., 2010), squamouscell carcinoma of the larynx (SCCL) (Cordes et al., 2010), oral squamouscell carcinomas (SCC) (Shigeishi et al., 2009), hepatocellularcarcinomas (HCC) (Satow et al., 2010), pancreatic tumor (Warner et al.,2009), ovarian cancer (Ramakrishna et al., 2010), squamous cellcarcinoma of the lung (Lin et al., 2006; Ma et al., 2006). It isfrequently co-overexpressed with Aurora-A giving rise to a novelfunctional unit with oncogenic properties (Asteriti et al., 2010). TPX2expression is a prognostic indicator in lung cancer (Kadara et al.,2009).

Hyaluronan-Mediated Motility Receptor (RHAMM) (HMMR)

The receptor for hyaluronan-mediated motility RHAMM (HMMR) exertsdifferent functions in the cell as well as on the cell membrane. RHAMMcan be exported to the cell surface where it binds hyaluronic acid (HA)and interacts with the HA receptor CD44. Processes like cell motility,wound healing and invasion are modulated by RHAMM (Sohr and Engeland,2008). RHAMM (receptor for HYA-mediating motility) is one of thereceptors for hyaluronan (HYA) (Gares and Pilarski, 2000). Also cancercells exhibit binding sites (CD44, RHAMM, etc.) for HYA and HYA protectscancer cells against immune cell attack. Serum HYA is often increased inmetastatic patients (Delpech et al., 1997). In addition, HYA-interactionwith RHAMM (HMMR) and CD44 on cancer cells has been proposed to beimportant in promoting tumor progression and dissemination (Li et al.,2000b). Furthermore, RHAMM is overexpressed in several cancer tissues(Tzankov et al., 2011); (Kramer et al., 2010); (Twarock et al., 2010);(Shigeishi et al., 2009); (Zlobec et al., 2008); (Li et al., 2000a)).

ADAM Metallopeptidase Domain 8 (ADAM8)

ADAM8 is a member of the ADAM (a disintegrin and metalloprotease domain)family. Many ADAM species, including ADAM8, are expressed in humanmalignant tumors, where they are involved in the regulation of growthfactor activities and integrin functions, leading to promotion of cellgrowth and invasion (Mochizuki and Okada, 2007). The expression of ADAM8was positively correlated to EGFR. Both were mainly expressed in thecytoplasm and on the cell membrane (Wu et al., 2008). ADAM8 wasabundantly expressed in the great majority of lung cancers examined.Exogenous expression of ADAM8 increased the migratory activity ofmammalian cells, an indication that ADAM8 may play a significant role inprogression of lung cancer (Ishikawa et al., 2004). ADAM8 has beenassociated with poor prognosis of lung cancer (Hemandez et al., 2010).ADAM8 over-expression was associated with shorter patient survival andit was a good predictor of distant metastases in RCC (Roemer et al.,2004b; Roemer et al., 2004a). In addition, expression levels and theprotease activities of ADAM8 correlated with invasive activity of gliomacells, indicating that ADAM8 may play a significant role in tumorinvasion in brain cancer (Wildeboer et al., 2006).

Collagen Alpha-3(VI) Chain Protein (COL6A3)

COL6A3 encodes the alpha-3 chain, one of the three alpha chains of typeVI collagen. The protein domains have been shown to bind extracellularmatrix proteins, an interaction that explains the importance of thiscollagen in organizing matrix components.

Remodeling of the extracellular matrix through overexpression ofcollagen VI contributes to cis-platin resistance in ovarian cancercells. The presence of collagen VI correlated with tumor grade, anovarian cancer prognostic factor (Sherman-Baust et al., 2003). COL6A3 isoverexpressed in colorectal tumor (Smith et al., 2009a), salivary glandcarcinoma (Leivo et al., 2005) and differentially expressed in gastriccancer (Yang et al., 2007). COL6A3 was identified as one of seven geneswith tumor-specific splice variants. The validated tumor-specificsplicing alterations were highly consistent, enabling clear separationof normal and cancer samples and in some cases even of different tumorstages (Thorsen et al., 2008).

Thy-1 Cell Surface Antigen (THY1)

Thy-1 (CD90) is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchoredglycol-protein expressed on many cell types, including T cells,thymocytes, neurons, endothelial cells, and fibroblasts. Activation ofThy-1 can promote T cell activation. Thy-1 also affects numerousnon-immunologic biological processes, including cellular adhesion,neurite outgrowth, tumor growth, tumor suppression, migration, woundhealing and cell death. Thy-1 is an important regulator of cell-cell andcell-matrix interactions, with important roles in nerve regeneration,metastasis, inflammation, and fibrosis (Rege and Hagood, 2006b; Rege andHagood, 2006a). Furthermore, Thy-1 appears to be a marker of adult butnot embryonic angiogenesis. The up-regulation of Thy-1 by cytokines butnot growth factors indicates the importance of inflammation in thepathogenesis of adult angiogenesis (Lee et al., 1998). There is asignificant overexpression of Thy-1 located in the lung cancer cellnucleus as compared to the normal tissue or benign tumor cells of lung,and it is one of the factors related to the prognosis of NSCLC patients.Thus, Thy-1 may be a novel latent malignant marker in the lung cancerpathology (Chen et al., 2005b). Thy-1 can be considered as a surrogatemarker for various kind of stem cells (mesenchymal stem cells, hepaticstem cells (“oval cells”) (Masson et al., 2006), keratinocyte stem cells(Nakamura et al., 2006) and hematopoietic stem cells (Yamazaki et al.,2009).

Deiodinase, Iodothyronine, Type II (DIO2)

The protein encoded by the DIO2 gene belongs to the iodothyroninedeiodinase family. It is highly expressed in the thyroid, and maycontribute significantly to the relative increase in thyroidal T3production in patients with Graves' disease and thyroid adenomas (Meyeret al., 2008); (de Souza Meyer et al., 2005)). The gene expressionpatterns are significantly different between upward and downwardprogressing types of nasopharyngeal carcinoma (NPC). The expression ofDIO2 gene is higher in the downward progressing type (downward=distantmetastasis) than in upward progressing type (local growth and invasionof the base of skull), which may be closely related to the metastasispotential of NPC (Liang et al., 2008). DIO2 mRNA as well as DIO2activity is expressed in brain tumors (Murakami et al., 2000). D2activity in lung is present and similar in peripheral lung and lungcancer tissue (Wawrzynska et al., 2003).

Periostin, Osteoblast Specific Factor (POSTN)

POSTN, a gene encoding a protein with similarity to the fasciclin familyand involved in cell survival and angiogenesis, has emerged as apromising marker for tumor progression in various types of human cancers(Ruan et al., 2009).

High expression of periostin protein or mRNA was detected in most solidtumors including breast (Zhang et al., 2010b), colon (Kikuchi et al.,2008), head and neck (Kudo et al., 2006), pancreatic (Kanno et al.,2008), papillary thyroid (Puppin et al., 2008), prostate (Tischler etal., 2010), ovarian (Choi et al., 2010), lung (Takanami et al., 2008)and liver (Utispan et al., 2010) carcinoma, as well as oesophagealsquamous cell carcinoma (Kwon et al., 2009). Periostin is abnormallyhighly expressed in lung cancer and is correlated with angiogenesis,invasion and metastasis (Takanami et al., 2008). Silencing of periostinin A549 non-small cell lung cancer (NSCLC) cells inhibits tumor cellgrowth and decrease cell invasion (Wu et al., 2013).

SLIT1 (Slit Homolog 1 (Drosophila)), SLIT2 (Slit Homolog 2 (Drosophila))

SLITs (SLIT1, SLIT2, and SLIT3) are a family of secreted proteins thatmediate positional interactions between cells and their environmentduring development by signaling through ROBO receptors (Hinck, 2004).SLIT/ROBO signaling, however, is not restricted to development, and lossof these cues likely plays an important role during tumor progression.Slits and Robos are considered candidate tumor suppressor genes becausetheir promoters are frequently hypermethylated in epithelial cancers(Narayan et al., 2006; Schmid et al., 2007; Latil et al., 2003). In ˜50%of sampled human breast tumors, SLIT2 or SLIT3 gene expression issilenced (Sharma et al., 2007). Hypermethylation of SLIT2 was frequentlydetected in NSCLCs and associated with various clinical features (Suzukiet al., 2013).

TLX3 (T-Cel Leukemia Homeobox 3)

TLX3 (also known as RNX or HOX11L2) belongs to a family of orphanhomeobox genes that encode DNA-binding nuclear transcription factors.Members of the HOX11 gene family are characterized by a threonine-47replacing cytosine in the highly conserved homeodomain (Dear et al.,1993). TLX3 is uniquely expressed in the developing medulla oblongataand is required for proper formation of first-order relay visceralsensory neurons and of most of the (nor) adrenergic centers in thebrainstem, especially involved in the physiologic control ofcardiovascular and respiratory systems (Qian et al., 2001). Expressionof TLX3 has also been detected in leukaemia samples from 20% of childrenand 13% of adults affected with T-cell acute lymphocytic leukaemia (Caveet al., 2004), although this gene has never been involved in normalT-cell differentiation (Ferrando et al., 2004).

CEP192 (Centrosomal Protein 192 kDa)

Centrosomes play an important role in various cellular processes,including spindle formation and chromosome segregation. CEP192 is acentrosome protein that plays a critical role in centrosome biogenesisand function in mammals, Drosophila and C. elegans (Gomez-Ferreria etal., 2012). It stimulates the formation of the scaffolding upon whichgamma tubulin ring complexes and other proteins involved in microtubulenucleation and spindle assembly become functional during mitosis(Gomez-Ferreria et al., 2007).

ANKS1A (Ankyrin Repeat and Sterile Alpha Motif Domain Containing 1A)

Ankyrin repeat and SAM domain-containing protein IA is a protein that inhumans is encoded by the ANKS1A gene (Nagase et al., 1996). ANKS1A hasbeen first described as a target and signal transmitter of receptortyrosine kinases like EGFR and PDGFR (Pandey et al., 2002) and morerecently as an interaction partner of the receptor tyrosine kinase EphA8(Shin et al., 2007). In a recent study, single-nucleotide polymorphisms(SNPs) were genotyped in 348 advanced NSCLC patients. They identified 17top candidate SNPs related to prognosis. SNPs were located in thegenomic region of the ANKS1A gene (Lee et al., 2013).

CEP250 (Centrosomal Protein 250 kDa)

The CEP250 gene encodes a core centrosomal protein required forcentriole-centriole cohesion during interphase of the cell cycle (Mayoret al., 2002). By radiation hybrid analysis, Fry et al. (1998) mappedthe CEP250 gene to the centromeric region of chromosome 20, atapproximately 20q11.2 (Fry et al., 1998). Mayor et al. (2002) found thatoverexpression of CEP250 in a human osteosarcoma cell line resulted information of large centrosome-associated structures. CEP250overexpression did not interfere with centrosome separation or celldivision, however, indicating that cell cycle-regulated activitydissociates CEP250 from centrosomes (Mayor et al., 2002).

MDN1 (MDN1, Midasin Homolog (Yeast))

MDN1, midasin homolog (yeast) is a protein that in humans is encoded bythe MDN1 gene. Midasin is present as a single-copy gene encoding awell-conserved protein of approximately 600 kDa in all eukaryotes forwhich data are available. In humans, the gene maps to 6q15 and encodes apredicted protein of 5596 residues (632 kDa) (Garbarino and Gibbons,2002). Recently, MDN1 was found to be mutated in breast cancers of theluminal B subtype. MDN1 may play a role in the development and hormoneresistance of this aggressive subtype (Comen et al., 2014).

OLFM1 (Olfactomedin 1)

OLFM1, also called Noelin-1, is a secreted glycoprotein belonging to afamily of olfactomedin domain-containing proteins and plays an importantrole in regulating the production of neural crest cells by the neuraltube (Barembaum et al., 2000). Olfactomedin was originally identified asthe major component of the mucus layer that surrounds the chemosensorydendrites of olfactory neurons (Kulkarni et al., 2000). Expression ofolfactomedin 1 protein was significantly higher in lung adenocarcinomathan in lung cancer of other histologic types and normal lung tissues(Wu et al., 2010). Furthermore, OLFM1 is deregulated in the endometrialcancer, Ewing's sarcoma, and neuroblastoma (Wong et al., 2007; Allanderet al., 2002; Khan et al., 2001).

BUB1B (Budding Uninhibited by Benzimidazoles 1 Homolog Beta (Yeast))

BUB1B, also named BubR1, is a core mitotic checkpoint component thatbinds to and inhibits the Cdc20-activated anaphase-promoting complex(APC/CCdc20), a ubiquitin E3 ligase that initiates anaphase byorchestrating separase-mediated cleavage of cohesion rings that holdsister chromatids together (Baker et al., 2004). BubR1 not onlycontributes to proper chromosome segregation through mitotic checkpointactivation but also by regulation of chromosome-spindle attachments(Malureanu et al., 2009; Lampson and Kapoor, 2005). Impaired spindlecheckpoint function has been found in many forms of cancer. Mutations inBubR1 have been associated with mosaic variegated aneuploidy (MVA), arare human syndrome characterized by aneuploidization, tumorpredisposition, and several progeroid traits, including short lifespan,growth and mental retardation, cataracts, and facial dysmorphisms(Matsuura et al., 2006).

PI4KA (Phosphatidylinositol 4-Kinase, Catalytic, Alpha)

Four different phosphatidylinositol 4-kinases (PI4Ks) are expressed inhuman cells. These isoenzymes (PI4KA, PI4 KB, PI4K2A and PI4K2B)catalyze the phosphorylation of phosphatidylinositol (PtdIns) in thecytoplasmic face of cellular membranes, leading to the production ofphosphatidylinositol 4-phosphate (PtdIns4P) (Minogue and Waugh, 2012).PI4KA is mainly found in the endoplasmic reticulum (ER). Its activityseems to regulate both the formation of ER exit sites (Blumental-Perryet al., 2006) and the concentration of PtdIns4P in the plasma membrane(Balla et al., 2008). A research group has found that PI4KA mRNA wasmore abundant in HCC than normal healthy tissues. This up-regulationcorrelated significantly with both poor differentiation and the activeproliferation rate in HCC. Therefore PI4KA could be used as a newmolecular marker to improve established prognostic models for HCC(Ilboudo et al., 2014).

AURKB (Aurora Kinase B)

Aurora B kinase is a protein that functions in the attachment of themitotic spindle to the centromere (Kim et al., 2011). AURKB localizes tomicrotubules near kinetochores (Kunitoku et al., 2003). Aurora kinasesare over-expressed in a variety of tumor cell lines, suggesting thatthese kinases might play a role in tumorigenesis, and have alreadybecome potential targets for cancer diagnosis and therapy (Fu et al.,2007). Recently a gene signature of five genes (TOP2A, AURKB, BRRN1,CDK1 and FUS) that are closely associated with the outcomes in patientswith NSCLC was identified. The results suggested that genes involved inchromosome condensation, like AURKB, are likely related with stem-likeproperties and might predict survival in lung adenocarcinoma (Perumal etal., 2012).

SLC3A2 (Solute Carrier Family 3 (Activators of Dibasic and Neutral AminoAcid Transport), Member 2)

SLC3A2 comprises the light subunit of the large neutral amino acidtransporter (LAT) that is also known as CD98 (cluster of differentiation98) (Lemaitre et al., 2005). The CD98 heterodimer consists of a type IIsingle-pass transmembrane heavy chain (CD98hc, also known as 4F2 antigenheavy chain or FRP-1; encoded by the genes SLC3A2 and Slc3a2 for humanand mouse, respectively) of ˜80-85 kDa that is disulfide-linked with amulti-pass light chain of ˜40 kDa (Deves and Boyd, 2000). CD98hcfunctions in amplifying integrin signalling and in the transport ofamino acids; both of these functions can contribute to cell survival andproliferation (Cantor and Ginsberg, 2012). Many tumors express CD98hc(SLC3A2), and its expression correlates with poor prognosis in B celllymphomas. Furthermore, nearly all studies that have examined theexpression of CD98hc or CD98 light chains in solid tumors show thattheir expression is correlated with progressive or metastatic tumors(Kaira et al., 2009).

IFT81 (Intraflagellar Transport 81 Homolog (Chlamydomonas))

Intraflagellar transport (IFT) of ciliary precursors such as tubulinfrom the cytoplasm to the ciliary tip is involved in the construction ofthe cilium, a hairlike organelle found on most eukaryotic cells.Knockdown of IFT81 and rescue experiments with point mutants showed thattubulin binding by IFT81 was required for ciliogenesis in human cells(Bhogaraju et al., 2013). Together with IFT74/72, IFT81 forms a corecomplex to build IFT particles which are required for cilium formation(Lucker et al., 2005).

COG4 (Component of Oligomeric Golgi Complex 4)

The COG complex consists of eight subunits named COG1-8 (Ungar et al.,2002; Whyte and Munro, 2001) grouped into two sub-complexes: COG1-4(Lobe A) and COG5-8 (Lobe B) (Ungar et al., 2005). The COG complexfunctions in the tethering of vesicles recycling resident Golgi proteins(such as glycosylation enzymes) (Pokrovskaya et al., 2011). The COG4gene maps to chromosome 16q22.1 (Reynders et al., 2009). Ungar et al.(2002) concluded that COG4 is critical for the structure and function ofthe Golgi apparatus and can influence intracellular membrane trafficking(Ungar et al., 2002).

NCBP1 (Nuclear Cap Binding Protein Subunit 1, 80 kDa)

Nuclear cap-binding protein complex is a RNA-binding protein which bindsto the 5′ cap of RNA polymerase II. Kataoka et al. (1994) described thecloning of a gene that encodes an 80-kD nuclear cap-binding protein(NCBP1) found in HeLa cell nuclear extracts that may be involved in mRNAsplicing and RNA export (Kataoka et al., 1994). By hybridizing togenomic DNA from a somatic cell hybrid panel, Chadwick et al. (1996)mapped the NCBP1 gene to 9q34.1 (Chadwick et al., 1996).

NEFH (Neurofilament, Heavy Polypeptide)

The NEFH encoding neurofilament heavy chain is one of the majorcomponents of the neuronal cytoskeleton neurofilaments. Theneurofilament heavy polypeptide (NEFH, 200 kD) gene resides atchromosomal band 22q12.2 and was proposed as a DNA marker forpresymptomatic diagnosis in neurofibromatosis type 2 (NF2) families.Loss or down-regulation of NEFH has been mostly reported in humanautonomic nerve tumors or central neurocytomas (Mena et al., 2001; Segalet al., 1994). In addition, absent or diminished NEFH expression inhuman prostate cancer (Schleicher et al., 1997), clear-cell epitheloidtumor (Tanaka et al., 2000), and small cell lung carcinoma (Bobos etal., 2006) has been observed. Interestingly, over-expression of NEFHdisrupted normal cell structure and function, and induced cell death(Szebenyi et al., 2002).

DETAILED DESCRIPTION OF THE INVENTION

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13 or 14 and in caseof MHC class II peptides they can be as long as 15, 16, 17, 18, 19 or 20amino acids in length.

Further the term “peptide” shall include salts of a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids.Preferably the salts are pharmaceutical acceptable salts.

The term “peptide” shall include “oligopeptide”. The term “oligopeptide”is used herein to designate a series of amino acid residues, connectedone to the other typically by peptide bonds between the alpha-amino andcarbonyl groups of the adjacent amino acids. The length of theoligopeptide is not critical to the invention, as long as the correctepitope or epitopes are maintained therein. The oligopeptides aretypically less than about 30 amino acid residues in length, and greaterthan about 15 amino acids in length.

The term “the peptides of the present invention” shall include thepeptides consisting of or comprising a peptide as defined aboveaccording to SEQ ID No. 1 to SEQ ID No. 92.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 2 Expression frequencies F of HLA*A02 and the most frequent HLA-DRserotypes. Frequencies are deduced from haplotype frequencies G_(f)within the American population adapted from Mori et al. (Mori et al.,1997) employing the Hardy-Weinberg formula F = 1 − (1 − G_(f))².Combinations of A*02 with certain HLA-DR alleles might be enriched orless frequent than expected from their single frequencies due to linkagedisequilibrium. For details refer to Chanock et al. (Chanock et al.,2004). Expression frequencies of HLA*02 and HLA-DR serotypes withinNorth American subpopulations Caucasian African HLA Allele AmericanAmerican Asian American Latin American A*02 49.1% 34.1% 43.2% 48.3% DR119.4% 13.2% 6.8% 15.3% DR2 28.2% 29.8% 33.8% 21.2% DR3 20.6% 24.8% 9.2%15.2% DR4 30.7% 11.1% 28.6% 36.8% DR5 23.3% 31.1% 30.0% 20.0% DR6 26.7%33.7% 25.1% 31.1% DR7 24.8% 19.2% 13.4% 20.2% DR8 5.7% 12.1% 12.7% 18.6%DR9 2.1% 5.8% 18.6% 2.1%

Therefore, for therapeutic and diagnostic purposes a peptide that bindswith appropriate affinity to several different HLA class II receptors ishighly desirable. A peptide binding to several different HLA class IImolecules is called a promiscuous binder.

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence. The term “codingregion” refers to that portion of a gene which either naturally ornormally codes for the expression product of that gene in its naturalgenomic environment, i.e., the region coding in vivo for the nativeexpression product of the gene.

The coding region can be from a non-mutated (“normal”), mutated oraltered gene, or can even be from a DNA sequence, or gene, whollysynthesized in the laboratory using methods well known to those of skillin the art of DNA synthesis.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding (or encoding) for apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is going to be expressed by.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes.

Sequences of non-translated DNA may be present downstream from the openreading frame, where the same do not interfere with manipulation orexpression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a rabbit or a mouse, and also including a human, such immuneresponse taking the form of stimulating a T-cell response within therecipient animal, such as a human. Alternatively, the “active fragment”may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment,” when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:

Percent Identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the Percent Identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum Percent Identity to theReference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.

Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gin); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would simultaneously be substituted.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4.

Combinations of the elongations according to the invention can bedepicted from table 3:

C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation can be the peptides of the originalsequence of the protein or any other amino acid. The elongation can beused to enhance the stability or solubility of the peptides.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted CTLs, effector functions may be lysisof peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

Preferably, when the CTLs specific for a peptide of SEQ ID No. 1 to SEQID No. 92 are tested against the substituted peptides, the peptideconcentration at which the substituted peptides achieve half the maximalincrease in lysis relative to background is no more than about 1 mM,preferably no more than about 1 μM, more preferably no more than about 1nM, and still more preferably no more than about 100 pM, and mostpreferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by CTLs from more than one individual,at least two, and more preferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than 4 residues from thereference peptide, as long as they have substantially identicalantigenic activity.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has now raised thepossibility of using a host's immune system to intervene in tumorgrowth. Various mechanisms of harnessing both the humoral and cellulararms of the immune system are currently explored for cancerimmunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofcytotoxic T-cells (CTL) from tumor-infiltrating cell populations or fromperipheral blood suggests that such cells play an important role innatural immune defences against cancer. CD8-positive T-cells inparticular, which recognize class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 12residues derived from proteins or defect ribosomal products (DRIPS)located in the cytosols, play an important role in this response. TheMHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

MHC class I molecules can be found on most cells having a nucleus whichpresent peptides that result from proteolytic cleavage of mainlyendogenous, cytosolic or nuclear proteins, DRIPS, and larger peptides.However, peptides derived from endosomal compartments or exogenoussources are also frequently found on MHC class I molecules. Thisnon-classical way of class I presentation is referred to ascross-presentation in literature.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by eitherCD8-positive CTLs (MHC class I molecule) or by CD4-positive CTLs (MHCclass II molecule) is important in the development of tumor vaccines. Itis therefore an object of the present invention, to provide compositionsof peptides that contain peptides binding to MHC complexes of eitherclass.

Considering the severe side-effects and expense associated with treatingcancer better prognosis and diagnostic methods are desperately needed.Therefore, there is a need to identify other factors representingbiomarkers for cancer in general and lung cancer in particular.Furthermore, there is a need to identify factors that can be used in thetreatment of cancer in general and lung cancer in particular.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably lung cancers, even more preferably non-smallcell lung carcinoma (NSCLC) that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanlung cancer samples (see example 1, and FIG. 1).

The source gene/protein (also designated “full-length protein” or“underlying protein”) from which the peptides are derived were shown tobe highly overexpressed in non-small cell lung carcinoma, and for SEQIDs No. 66 to 75 gastric cancer and glioblastoma compared with normaltissues (see example 2, and FIG. 2 for NSCLC) demonstrating a highdegree of tumor association of the source genes. Moreover, the peptidesthemselves are strongly over-presented on tumor tissue but not on normaltissues (see example 3 and FIG. 3).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes/T cells. T cells can destroy the cells presenting therecognized HLA/peptide complex, e.g. lung cancer cells presenting thederived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention (see example 4 and FIG. 4).

Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt. As usedherein, “a pharmaceutically acceptable salt” refers to a derivative ofthe disclosed peptides wherein the peptide is modified by making acid orbase salts of the agent. For example, acid salts are prepared from thefree base (typically wherein the neutral form of the drug has a neutral—NH₂ group) involving reaction with a suitable acid. Suitable acids forpreparing acid salts include both organic acids, e.g., acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, salicylicacid, and the like, as well as inorganic acids, e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and thelike. Conversely, preparation of basic salts of acid moieties which maybe present on a peptide are prepared using a pharmaceutically acceptablebase such as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine or the like.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from lung cancer cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies can assist apathologist in diagnosis of cancer. Detection of certain peptides bymeans of antibodies, mass spectrometry or other methods known in the artcan tell the pathologist that the tissue is malignant or inflamed orgenerally diseased. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate markers in immunotherapy approaches aiming to inducelymphocyte responses by different means, e.g. vaccination of protein,nucleic acids, autologous materials, adoptive transfer of lymphocytes.

In gene therapy settings, lymphocyte responses against peptides can beconsidered in the assessment of side effects. Monitoring of lymphocyteresponses might also be a valuable tool for follow-up examinations oftransplantation therapies, e.g. for the detection of graft versus hostand host versus graft diseases.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bispecific antibody and/or a chimeric antibody.

Yet another aspect of the present invention then relates to a method ofproducing said antibody specifically binding to a human majorhistocompatibility complex (MHC) class I or II being complexed with aHLA-restricted antigen, the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically bindable to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen. Respective methods for producing such antibodiesand single chain class I major histocompatibility complexes, as well asother tools for the production of these antibodies are disclosed in WO03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and Cohen C J,Denkberg G, Lev A, Epel M, Reiter Y. Recombinant antibodies withMHC-restricted, peptide-specific, T-cell receptor-like specificity: newtools to study antigen presentation and TCR-peptide-MHC interactions. JMol Recognit. 2003 September-October; 16(5):324-32; Denkberg G, Lev A,Eisenbach L, Benhar I, Reiter Y. Selective targeting of melanoma andAPCs using a recombinant antibody with TCR-like specificity directedtoward a melanoma differentiation antigen. J Immunol. 2003 Sep. 1;171(5):2197-207; and Cohen C J, Sarig O, Yamano Y, Tomaru U, Jacobson S,Reiter Y. Direct phenotypic analysis of human MHC class I antigenpresentation: visualization, quantitation, and in situ detection ofhuman viral epitopes using peptide-specific, MHC-restricted humanrecombinant antibodies. J Immunol. 2003 Apr. 15; 170(8):4349-61, whichfor the purposes of the present invention are all explicitlyincorporated by reference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isregarded as “specific” in the context of the present invention.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor recognizing a specific peptide-MHCcomplex. Such soluble T-cell receptors can be generated from specificT-cell clones, and their affinity can be increased by mutagenesistargeting the complementarity-determining regions. For the purpose ofT-cell receptor selection, phage display can be used (US 2010/0113300,Liddy N, Bossi G, Adams K J, Lissina A, Mahon T M, Hassan N J, et al.Monoclonal TCR-redirected tumor cell killing. Nat Med 2012 June;18(6):980-987). For the purpose of stabilization of T-cell receptorsduring phage display and in case of practical use as drug, alpha andbeta chain can be linked e.g. by non-native disulfide bonds, othercovalent bonds (single-chain T-cell receptor), or by dimerizationdomains (see Boulter J M, Glick M, Todorov P T, Baston E, Sami M,Rizkallah P, et al. Stable, soluble T-cell receptor molecules forcrystallization and therapeutics. Protein Eng 2003 September;16(9):707-711; Card K F, Price-Schiavi S A, Liu B, Thomson E, Nieves E,Belmont H, et al. A soluble single-chain T-cell receptor IL-2 fusionprotein retains MHC-restricted peptide specificity and IL-2 bioactivity.Cancer Immunol Immunother 2004 April; 53(4):345-357; and Willcox B E,Gao G F, Wyer J R, O'Callaghan C A, Boulter J M, Jones E Y, et al.Production of soluble alphabeta T-cell receptor heterodimers suitablefor biophysical analysis of ligand binding. Protein Sci 1999 November; 8(11):2418-2423). The T-cell receptor can be linked to toxins, drugs,cytokines (see US 2013/0115191), domains recruiting effector cells suchas an anti-CD3 domain, etc., in order to execute particular functions ontarget cells. Moreover, it could be expressed in T cells used foradoptive transfer.

Further information can be found in WO 2004/033685A1 and WO2004/074322A1. A combination of sTCRs is described in WO 2012/056407A1.Further methods for the production are disclosed in WO 2013/057586A1.

In addition, they can be used to verify a pathologist's diagnosis of acancer based on a biopsied sample.

To select over-presented peptides, a presentation profile is calculatedshowing the median sample presentation as well as replicate variation.The profile juxtaposes samples of the tumor entity of interest to abaseline of normal tissue samples. Each of these profiles can then beconsolidated into an over-presentation score by calculating the p-valueof a Linear Mixed-Effects Model (J. Pinheiro, D. Bates, S. DebRoy,Sarkar D., R Core team. nlme: Linear and Nonlinear Mixed Effects Models.2008) adjusting for multiple testing by False Discovery Rate (Y.Benjamini and Y. Hochberg. Controlling the False Discovery Rate: APractical and Powerful Approach to Multiple Testing. Journal of theRoyal Statistical Society. Series B (Methodological), Vol. 57 (No.1):289-300, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from NSCLC samples with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary tumortissue obtained from NSCLC patients.

The proprietary discovery pipeline XPRESIDENT® v2.1 (see, for example,US 2013-0096016 which is hereby incorporated in its entirety) allows theidentification and selection of relevant over-presented peptide vaccinecandidates based on direct relative quantitation of HLA-restrictedpeptide levels on cancer tissues in comparison to several differentnon-cancerous tissues and organs. This was achieved by the developmentof label-free differential quantitation using the acquired LC-MS dataprocessed by a proprietary data analysis pipeline, combining algorithmsfor sequence identification, spectral clustering, ion counting,retention time alignment, charge state deconvolution and normalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from 50 shock-frozen NSCLC tumor tissue sampleswere purified and HLA-associated peptides were isolated and analysed byLC-MS.

All TUMAPs contained in the application at hand were identified withthis approach on primary NSCLC tumor samples confirming theirpresentation on primary NSCLC.

TUMAPs identified on multiple NSCLC tumor and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

The present invention therefore relates to a peptide comprising asequence that is selected from the group consisting of SEQ ID No. 1 toSEQ ID No. 65, and SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92 ora variant thereof which is at least 90% homologous (preferablyidentical) to SEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ IDNo. 84 and SEQ ID No. 92 or a variant thereof that induces T cellscross-reacting with said peptide, wherein said peptide is not afull-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID No. 1 to SEQ ID No.65 and SEQ IDs No. 76 to SEQ ID No. 84, and SEQ ID No. 92 or a variantthereof which is at least 90% homologous (preferably identical) to SEQID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ ID No. 84, whereinsaid peptide or variant has an overall length of between 8 and 100,preferably between 8 and 30, and most preferred between 8 and 14 aminoacids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID No. 1 to SEQ ID No. 65, and SEQID No. 76 to SEQ ID No. 84, and SEQ ID No. 92.

The present invention further relates to the peptides according to theinvention, wherein the peptide is modified and/or includes non-peptidebonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is a fusion protein, in particularcomprising N-terminal amino acids of the HLA-DR antigen-associatedinvariant chain (Ii), or wherein the peptide is fused to (or into) anantibody, such as, for example, an antibody that is specific fordendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe full human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the invention.

The present invention further relates to a peptide according to theinvention, a nucleic acid according to the invention or an expressionvector according to the invention for use in medicine.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to theinvention that is an antigen presenting cell.

The present invention further relates to the host cell according to theinvention wherein the antigen presenting cell is a dendritic cell.

The present invention further relates to a method of producing a peptideaccording to the invention, the method comprising culturing the hostcell described and isolating the peptide from the host cell or itsculture medium.

The present invention further relates to an in vitro method forproducing activated cytotoxic T lymphocytes (CTL), the method comprisingcontacting in vitro CTL with antigen loaded human class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellfor a period of time sufficient to activate said CTL in an antigenspecific manner, wherein said antigen is any peptide according to theinvention.

The present invention further relates to the method as described,wherein said antigen is loaded onto class I or II MHC moleculesexpressed on the surface of a suitable antigen-presenting cell bycontacting a sufficient amount of the antigen with an antigen-presentingcell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID No. 1 to SEQID No. 65, and SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92 or saidvariant amino acid sequence.

The present invention further relates to activated cytotoxic Tlymphocytes (CTL), produced by the method according to the invention,which selectively recognise a cell which aberrantly expresses apolypeptide comprising an amino acid sequence described.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the invention, themethod comprising administering to the patient an effective number ofcytotoxic T lymphocytes (CTL) according to the invention.

The present invention further relates to the use of any peptideaccording to the invention, a nucleic acid according to the invention,an expression vector according to the invention, a cell according to theinvention, or an activated cytotoxic T lymphocyte according to theinvention as a medicament or in the manufacture of a medicament.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine.

The present invention further relates to a use according to theinvention, wherein the medicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are lung cancer cells, gastric,gastrointestinal, colorectal, pancreatic or renal.

The present invention further relates to particular marker proteins andbiomarkers that can be used in the prognosis of lung cancer.

Further, the present invention relates to the use of the novel targetsas described in accordance with the present invention for cancertreatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments or polymers of those immunoglobulin moleculesand humanized versions of immunoglobulin molecules, so long as theyexhibit any of the desired properties (e.g., specific binding of an lungcancer marker polypeptide, delivery of a toxin to an lung cancer cellexpressing a lung cancer marker gene at an increased level, and/orinhibiting the activity of a lung cancer marker polypeptide) accordingto the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length lung cancer marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques.

For example, a cDNA encoding a ABCA13, MMP12, DST, MXRA5, CDK4, HNRNPH,TANC2, 1RNF213, SMYD3 and SLC34A2, or any other polypeptide of SEQ IDNo. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ ID No. 84 and SEQ IDNo. 92 polypeptide, or a fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the lung cancer marker polypeptideused to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988, new 2^(nd) edition 2013). For example, the antibodies may betested in ELISA assays, Western blots, immunohistochemical staining offormalin-fixed lung cancers or frozen tissue sections. After theirinitial in vitro characterization, antibodies intended for therapeuticor in vivo diagnostic use are tested according to known clinical testingmethods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated by reference in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fe fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.

Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody for treating lung cancer, the efficacy ofthe therapeutic antibody can be assessed in various ways well known tothe skilled practitioner. For instance, the size, number, and/ordistribution of lung cancer in a subject receiving treatment may bemonitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment oflung cancer.

Because the lung tumor markers ABCA13, MMP12 of the invention are highlyexpressed in lung cancer cells and are expressed at extremely low levelsin normal cells, inhibition of ABCA13 and MMP12 expression orpolypeptide activity may be integrated into any therapeutic strategy fortreating or preventing NSCLC.

The principle of antisense therapy is based on the hypothesis thatsequence-specific suppression of gene expression (via transcription ortranslation) may be achieved by intra-cellular hybridization betweengenomic DNA or mRNA and a complementary antisense species. The formationof such a hybrid nucleic acid duplex interferes with transcription ofthe target tumor antigen-encoding genomic DNA, orprocessing/transport/translation and/or stability of the target tumorantigen mRNA.

Antisense nucleic acids can be delivered by a variety of approaches. Forexample, antisense oligonucleotides or anti-sense RNA can be directlyadministered (e.g., by intravenous injection) to a subject in a formthat allows uptake into tumor cells. Alternatively, viral or plasmidvectors that encode antisense RNA (or RNA fragments) can be introducedinto cells in vivo. Antisense effects can also be induced by sensesequences; however, the extent of phenotypic changes is highly variable.Phenotypic changes induced by effective antisense therapy are assessedaccording to changes in, e.g., target mRNA levels, target proteinlevels, and/or target protein activity levels.

In a specific example, inhibition of lung tumor marker function byantisense gene therapy may be accomplished by direct administration ofantisense lung tumor marker RNA to a subject. The antisense tumor markerRNA may be produced and isolated by any standard technique, but is mostreadily produced by in vitro transcription using an antisense tumormarker cDNA under the control of a high efficiency promoter (e.g., theT7 promoter). Administration of anti-sense tumor marker RNA to cells canbe carried out by any of the methods for direct nucleic acidadministration described below.

An alternative strategy for inhibiting ABCA13, and MMP12 function usinggene therapy involves intracellular expression of an anti-ABCA13, MMP12antibody or a portion of an anti-ABCA13, MMP12 antibody. For example,the gene (or gene fragment) encoding a monoclonal antibody thatspecifically binds to an ABCA13, MMP12 polypeptide and inhibits itsbiological activity is placed under the transcriptional control of aspecific (e.g., tissue- or tumor-specific) gene regulatory sequence,within a nucleic acid expression vector. The vector is then administeredto the subject such that it is taken up by lung cancer cells or othercells, which then secrete the anti-ABCA13, MMP12 antibody and therebyblock biological activity of the ABCA13, MMP12 polypeptide. Preferably,the ABCA13, MMP12 polypeptides are present at the extracellular surfaceof gastric cancer cells.

In the methods described above, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for inhibition ofgastric tumor marker protein expression. The vector can be acommercially available preparation, such as an adenovirus vector(Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of thenucleic acid or vector to cells can be via a variety of mechanisms. Asone example, delivery can be via a liposome, using commerciallyavailable liposome preparations such as LIPOFECTIN, LIPOFECTAMINE(GIBCO-25 BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden,Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as wellas other liposomes developed according to procedures standard in theart. In addition, the nucleic acid or vector of this invention can bedelivered in vivo by electroporation, the technology for which isavailable from Genetronics, Inc. (San Diego, Calif.) as well as by meansof a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system that can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells antisense nucleic acid thatinhibits expression of ABCA13, MMP12. The exact method of introducingthe altered nucleic acid into mammalian cells is, of course, not limitedto the use of retroviral vectors. Other techniques are widely availablefor this procedure including the use of adenoviral vectors,adeno-associated viral (AAV) vectors, lentiviral vectors, pseudotypedretroviral vectors. Physical transduction techniques can also be used,such as liposome delivery and receptor-mediated and other endocytosismechanisms. This invention can be used in conjunction with any of theseor other commonly used gene transfer methods.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moreABCA13, MMP12 targets and the affinity value (Kd) is less than 1×10 μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect the ABCA13,MMP12 proteins express in situ.

The present invention thus provides a peptide comprising a sequence thatis selected from the group of consisting of SEQ ID No. 1 to SEQ ID No.65, and SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92 or a variantthereof which is 90% homologous to SEQ ID No. 1 to SEQ ID No. 65, andSEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No. 92 or a variant thereofthat will induce T cells cross-reacting with said peptide.

The peptides of the invention have the ability to bind to a molecule ofthe human major histocompatibility complex (MHC) class-I and/or classII.

In the present invention, the term “homologous” refers to the degree ofidentity (see Percent Identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or otheranalysis tools are provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Fong et al., 2001); (Zaremba et al., 1997;Colombetti et al., 2006; Appay et al., 2006).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID No. 1 to SEQ ID No. 65, and SEQ IDNo. 76 to SEQ ID No. 84, and SEQ ID No. 92. For example, a peptide maybe modified so that it at least maintains, if not improves, the abilityto interact with and bind to the binding groove of a suitable MHCmolecule, such as HLA-A*02 or -DR, and in that way it at leastmaintains, if not improves, the ability to bind to the TCR of activatedCTL.

These CTL can subsequently cross-react with cells and kill cells thatexpress a polypeptide that contains the natural amino acid sequence ofthe cognate peptide as defined in the aspects of the invention. As canbe derived from the scientific literature (Rammensee et al., 1997) anddatabases (Rammensee et al., 1999), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID No.1 to SEQ ID No. 65 and SEQ ID No. 76 to SEQ ID No. 84, and SEQ ID No.92, by maintaining the known anchor residues, and would be able todetermine whether such variants maintain the ability to bind MHC class Ior II molecules. The variants of the present invention retain theability to bind to the TCR of activated CTL, which can subsequentlycross-react with- and kill cells that express a polypeptide containingthe natural amino acid sequence of the cognate peptide as defined in theaspects of the invention.

Those amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith another amino acid whose incorporation does not substantiallyaffect T-cell reactivity and does not eliminate binding to the relevantMHC. Thus, apart from the proviso given, the peptide of the inventionmay be any peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 4 Variants and motif of the peptides according to SEQ ID NO: 1, 2,4, 5 and 7 Position 1 2 3 4 5 6 7 8 9 ABCA13-001 Peptide Code I L F E IN P K L SEQ ID No. Variants V I A M V M I M M A A V A I A A A V V V I VV A T V T I T T A Q V Q I Q Q A MMP12-003 Peptide Code K I Q E M Q H F LSEQ ID No. Variants L V L I L L A M V M I M M A A V A I A A A V V V I VV A T V T I T T A Q V Q I Q DST-001 Peptide Code N L I E K S I Y L SEQID No. Variants V I A M V M I M M A A V A I A A A V V V I V V A T V T IT T A Q V Q I Q Q A MXRA5-001 Peptide Code T L S S I K V E V SEQ ID No.Variants I L A M M I M L M A A A I A L A A V V I V L V A T T I T L T A QQ I Q L Q A CDK4-001 Peptide Code T L W Y R A P E V SEQ ID No. VariantsI L A M M I M L M A A A I A L A A V V I V L V A T T I T L T A Q Q I Q LQ A

Longer peptides may also be suitable. It is also possible, that MHCclass I epitopes, although usually between 8-11 amino acids long, aregenerated by peptide processing from longer peptides or proteins thatinclude the actual epitope. It is preferred that the residues that flankthe actual epitope are residues that do not substantially affectproteolytic cleavage necessary to expose the actual epitope duringprocessing.

Accordingly, the present invention also provides peptides and variantsof MHC class I epitopes wherein the peptide or variant has an overalllength of between 8 and 100, preferably between 8 and 30, and mostpreferred between 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids,in case of the class II binding peptides the length can also be 15, 16,17, 18, 19, 20, 21 or 33 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I. Binding of a peptide or avariant to a MHC complex may be tested by methods known in the art.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ ID No. 84, andSEQ ID No. 92.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 to SEQ ID No. 84, and SEQID No. 92 or a variant thereof contains additional N- and/orC-terminally located stretches of amino acids that are not necessarilyforming part of the peptide that functions as an epitope for MHCmolecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is afusion protein which comprises, for example, the 80 N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (p33, in thefollowing “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that for MHCbinding and T helper cell responses, these pseudopeptides are useful.Retro-inverse peptides, which contain NH—CO bonds instead of CO—NHpeptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals.

Woodward's Reagent K may be used to modify specific glutamic acidresidues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be usedto form intra-molecular crosslinks between a lysine residue and aglutamic acid residue.

For example, diethylpyrocarbonate is a reagent for the modification ofhistidyl residues in proteins. Histidine can also be modified using4-hydroxy-2-nonenal.

The reaction of lysine residues and other α-amino groups is, forexample, useful in binding of peptides to surfaces or the cross-linkingof proteins/peptides. Lysine is the site of attachment ofpoly(ethylene)glycol and the major site of modification in theglycosylation of proteins.

Methionine residues in proteins can be modified with e.g. iodoacetamide,bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethyleneglycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamoylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lu et al (1981)and references therein. Temporary N-amino group protection is affordedby the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage ofthis highly base-labile protecting group is done using 20% piperidine inN, N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclo-hexyl-carbodiimide/hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example (Bruckdorfer et al., 2004) and the references as citedtherein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilisation of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by (Saikiet al., 1988)). This method may be used for introducing the DNA into asuitable vector, for example by engineering in suitable restrictionsites, or it may be used to modify the DNA in other useful ways as isknown in the art. If viral vectors are used, pox- or adenovirus vectorsare preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063,4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the fl origin. Vectorscontaining the preprotrypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.Acad. Sci. USA 69, 2110, and Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Transformation of yeast cells is described in Sherman et al (1986)Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y.The method of Beggs (1978) Nature 275, 104-109 is also useful. Withregard to vertebrate cells, reagents useful in transfecting such cells,for example calcium phosphate and DEAE-dextran or liposome formulations,are available from Stratagene Cloning Systems, or Life TechnologiesInc., Gaithersburg, Md. 20877, USA. Electroporation is also useful fortransforming and/or transfecting cells and is well known in the art fortransforming yeast cell, bacterial cells, insect cells and vertebratecells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) are currently under investigation for the treatment of prostatecancer (Sipuleucel-T) (Small et al., 2006; Rini et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al Nature Medicine 18, 1254-1261 (2012)).

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre et al 1985.

Preferably, the host cell before transfection expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7. 1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive CTLs.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No. 76 toSEQ ID No. 84, and SEQ ID No. 92, or a variant amino acid sequencethereof.

A number of other methods may be used for generating CTL in vitro. Forexample, the methods described in Peoples et al (1995) and Kawakami etal (1992) use autologous tumor-infiltrating lymphocytes in thegeneration of CTL. Plebanski et al (1995) makes use of autologousperipheral blood lymphocytes (PLBs) in the preparation of CTL. Jochmuset al (1997) describes the production of autologous CTL by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus. Hill et al (1995) and Jerome et al (1993) make use ofB cells in the production of autologous CTL. In addition, macrophagespulsed with peptide or polypeptide, or infected with recombinant virus,may be used in the preparation of autologous CTL. S. Walter et al. 2003describe the in vitro priming of T cells by using artificial antigenpresenting cells (aAPCs), which is also a suitable way for generating Tcells against the peptide of choice. In this study, aAPCs were generatedby the coupling of preformed MHC:peptide complexes to the surface ofpolystyrene particles (microbeads) by biotin:streptavidin biochemistry.This system permits the exact control of the MHC density on aAPCs, whichallows to selectively elicit high- or low-avidity antigen-specific Tcell responses with high efficiency from blood samples. Apart fromMHC:peptide complexes, aAPCs should carry other proteins withco-stimulatory activity like anti-CD28 antibodies coupled to theirsurface. Furthermore such aAPC-based systems often require the additionof appropriate soluble factors, e. g. cytokines like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al (1994)) which describes the development of cowpea mosaicvirus as a high-yielding system for the presentation of foreignpeptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID No. 1 to SEQ ID No. 92,preferably a sequence of SEQ ID No. 1 to SEQ ID No. 65, and SEQ ID No.76 to SEQ ID No. 84, and SEQ ID No. 92.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in (Gattinoni et al., 2006) and (Morganet al., 2006).

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated CTL, T-cell receptor or the nucleicacid encoding it is useful for the treatment of disorders, characterizedby cells escaping an immune response. Therefore any molecule of thepresent invention may be used as medicament or in the manufacture of amedicament. The molecule may be used by itself or combined with othermolecule(s) of the invention or (a) known molecule(s).

Preferably, the medicament of the present invention is a vaccine. It maybe administered directly into the patient, into the affected organ orsystemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo tocells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation of immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker, 1993). The peptide mayalso be tagged, may be a fusion protein, or may be a hybrid molecule.The peptides whose sequence is given in the present invention areexpected to stimulate CD4 or CD8 T cells. However, stimulation of CD8CTLs is more efficient in the presence of help provided by CD4 T-helpercells. Thus, for MHC Class I epitopes that stimulate CD8 CTL the fusionpartner or sections of a hybrid molecule suitably provide epitopes whichstimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes arewell known in the art and include those identified in the presentinvention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 92 and at leastone additional peptide, preferably two to 50, more preferably two to 25,even more preferably two to 20 and most preferably two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen or eighteen peptides. The peptide(s) may bederived from one or more specific TAAs and may bind to MHC class Imolecules.

In another aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 65 and SEQ IDNo. 76 to SEQ ID No. 84, and SEQ ID No. 92, and at least one additionalpeptide, preferably two to 50, more preferably two to 25, even morepreferably two to 20 and most preferably two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen or eighteen peptides. The peptide(s) may be derivedfrom one or more specific TAAs and may bind to MHC class I molecules.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNAor a combination thereof. Methods for designing and introducing such anucleic acid are well known in the art. An overview is provided by e.g.(Pascolo et al., 2005). Polynucleotide vaccines are easy to prepare, butthe mode of action of these vectors in inducing an immune response isnot fully understood. Suitable vectors and delivery systems includeviral DNA and/or RNA, such as systems based on adenovirus, vacciniavirus, retroviruses, herpes virus, adeno-associated virus or hybridscontaining elements of more than one virus. Non-viral delivery systemsinclude cationic lipids and cationic polymers and are well known in theart of DNA delivery. Physical delivery, such as via a “gene-gun,” mayalso be used. The peptide or peptides encoded by the nucleic acid may bea fusion protein, for example with an epitope that stimulates T cellsfor the respective opposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CTLs and helper-T(TH) cells to an antigen, and would thus be considered useful in themedicament of the present invention. Suitable adjuvants include, but arenot limited to, 1018 ISS, aluminium salts, AMPLIVAX®, AS15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived fromflagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA®),resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13, IL-21,Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch,ISS, ISCOMATRIX, IS-COMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995; Allison and Krummel, 1995). Also cytokines may be used.Several cytokines have been directly linked to influencing dendriticcell migration to lymphoid tissues (e.g., TNF-), accelerating thematuration of dendritic cells into efficient antigen-presenting cellsfor T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha. IFN-beta) (Gabrilovich, 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of T_(H1) cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The T_(H1) biasinduced by TLR9 stimulation is maintained even in the presence ofvaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA)that normally promote a T_(H2) bias. CpG oligonucleotides show evengreater adjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides andderivates, poly-(I:C) and derivates, RNA, sildenafil, and particulateformulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod.

In a preferred embodiment of the pharmaceutical composition according tothe invention, the adjuvant is cyclophosphamide, imiquimod orresiquimod.

Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40mAB or combinations thereof

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavours, lubricants, etc.The peptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients, 3^(rd) Ed., 2000, AmericanPharmaceutical Association and pharmaceutical press. The composition canbe used for a prevention, prophylaxis and/or therapy of adenomatous orcancerous diseases. Exemplary formulations can be found in, for example,EP2113253.

Nevertheless depending on the number and the physico-chemicalcharacteristics of the peptides of the invention further research isneeded to provide formulations for specific combinations of peptides,especially combinations with more than 20 peptides that are stable formore than 12 to 18 months.

The present invention provides a medicament that useful in treatingcancer, in particular non-small cell lung carcinoma, gastric cancer,renal cell carcinoma, colon cancer, adenocarcinoma, prostate cancer,benign neoplasm and malignant melanoma.

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceuticalcomposition thereof. The components of the kit may be pre-complexed oreach component may be in a separate distinct container prior toadministration to a patient. The components of the kit may be providedin one or more liquid solutions, preferably, an aqueous solution, morepreferably, a sterile aqueous solution. The components of the kit mayalso be provided as solids, which may be converted into liquids byaddition of suitable solvents, which are preferably provided in anotherdistinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably the administration is s.c., and most preferablyi.d. Administration may be by infusion pump.

Since the peptides of the invention were isolated from NSCLC, themedicament of the invention is preferably used to treat NSCLC. In apreferred embodiment, since the peptides of the invention derived fromABCA13 and MMP12 were isolated from NSCLC, the medicament of theinvention is preferably used to treat NSCLC.

The peptides with the SEQ ID Nos. 78 to 92 were also isolated fromMerkel cell carcinoma, and thus can be used to treat Merkel cellcarcinoma.

The present invention will now be described in the following examplesthat describe preferred embodiments thereof, nevertheless, without beinglimited thereto. For the purposes of the present invention, allreferences as cited herein are incorporated by reference in theirentireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Exemplary mass spectrum from ABCA13-001 demonstrating itspresentation on primary tumor sample NSCLC898. NanoESI-LCMS wasperformed on a peptide pool eluted from the NSCLC sample 898. The masschromatogram for m/z 543.8318±0.001 Da, z=2 shows a peptide peak at theretention time 86.36 min. B) The detected peak in the mass chromatogramat 86.36 min revealed a signal of m/z 543.8318 in the MS spectrum. C) Acollisionally induced decay mass spectrum from the selected precursorm/z 543.8318 recorded in the nanoESI-LCMS experiment at the givenretention time confirmed the presence of ABCA13-001 in the NSCLC898tumor sample. D) The fragmentation pattern of the synthetic ABCA13-001reference peptide was recorded and compared to the generated naturalTUMAP fragmentation pattern shown in C for sequence verification.

FIG. 2: Expression profiles of mRNA of selected proteins in normaltissues and in 21 lung cancer samples

a) ABCA13 (Probeset ID: 1553605_a_at)

b) MMP12 (Probeset ID: 204580_at)

FIG. 3: Presentation profiles for selected HLA class I peptides. Apresentation profile was calculated for each peptide showing the meansample presentation as well as replicate variations. The profilejuxtaposes samples of the tumor entity of interest to a baseline ofnormal tissue samples.

a) ABCA13-001 b) DST-001 c) MXRA5-001

FIG. 4: Exemplary results of peptide-specific in vitro immunogenicity ofclass I TUMAPs. Specific CD8+ T cells were stained with HLA multimerslinked to two different fluorochromes. Dot plots show the MHCmultimer-double-positive populations for the stimulating peptide (leftpanels) and the respective negative control stimulation (right panels).

FIG. 5: Binding properties of POSTN-002 and MMP12-002 to theinvestigated HLA haplotypes: The diagram shows the binding scores ofPOSTN-002 and MMP12-002 to 5 of the 7 analyzed HLA-DR haplotypes.

FIG. 6: Stability of HLA-POSTN-002 and MMP12-002 complexes after 24 h at37° C.: The diagram shows the percentage of intact HLA-POSTN-002 andHLA-MMP12-002 complexes after 24 h at 37° C. with a corresponding HLAmolecule.

FIG. 7: Exemplary vaccine-induced CD4 T-cell response to CEA-006 inclass II ICS assay. Following in vitro sensitization PBMCs of patient36-031 were analyzed for CD4 T-cell responses to CEA-006 (upper panel)and mock (lower panel) at time point pool V8/EOS. Cells were stimulatedwith corresponding peptides and stained with viability, anti-CD3,anti-CD8, anti-CD4 and effector markers (from right to left: CD154,TNF-alpha, IFN-gamma, IL-2, IL-10), respectively. Viable CD4 T cellswere analyzed for the proportion of cells positive for one or moreeffector molecules.

FIG. 8: Immunogenicity of various class II peptides: The diagram showsthe immune response rate to 5 various class II peptides detected in 16patients for IMA950 peptides and in 71 patients for IMA910 peptidesusing ICS.

EXAMPLES Example 1 Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface Tissue Samples

Patients' tumor tissues were provided by University of Heidelberg,Heidelberg, Germany. Written informed consents of all patients had beengiven before surgery. Tissues were shock-frozen in liquid nitrogenimmediately after surgery and stored until isolation of TUMAPs at −80°C.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk, K., 1991; Seeger, F. H. T., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, -C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Methods

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (Acquity UPLC system,Waters) and the eluting peptides were analyzed in an LTQ-Orbitrap hybridmass spectrometer (ThermoElectron) equipped with an ESI source. Peptidepools were loaded directly onto the analytical fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 1.7 μm C18reversed-phase material (Waters) applying a flow rate of 400 nL perminute. Subsequently, the peptides were separated using a two-step 180minute-binary gradient from 10% to 33% B at a flow rate of 300 nL perminute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometer was operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30.000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide. FIG. 1 shows an exemplary spectrum obtained fromtumor tissue for the MHC class I associated peptide ABCA13-001 and itselution profile on the UPLC system.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.2007a). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al. 2007b; Sturm et al. 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofile juxtaposes NSCLC samples to a baseline of normal tissue samples.

Presentation profiles of exemplary over-presented peptides are shown inFIG. 3.

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Not all peptides identified as being presented on the surface of tumorcells by MHC molecules are suitable for immunotherapy, because themajority of these peptides are derived from normal cellular proteinsexpressed by many cell types. Only few of these peptides aretumor-associated and likely able to induce T cells with a highspecificity of recognition for the tumor from which they were derived.In order to identify such peptides and minimize the risk forautoimmunity induced by vaccination the inventors focused on thosepeptides that are derived from proteins that are over-expressed on tumorcells compared to the majority of normal tissues.

The ideal peptide will be derived from a protein that is unique to thetumor and not present in any other tissue. To identify peptides that arederived from genes with an expression profile similar to the ideal onethe identified peptides were assigned to the proteins and genes,respectively, from which they were derived and expression profiles ofthese genes were generated.

RNA Sources and Preparation

Surgically removed tissue specimens were provided by University ofHeidelberg, Heidelberg, Germany (see Example 1) after written informedconsent had been obtained from each patient. Tumor tissue specimens weresnap-frozen in liquid nitrogen immediately after surgery and laterhomogenized with mortar and pestle under liquid nitrogen. Total RNA wasprepared from these samples using TRI Reagent (Ambion, Darmstadt,Germany) followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany);both methods were performed according to the manufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalisation, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed innon-small-cell lung carcinoma are shown in FIG. 2.

Example 4 In Vitro Immunogenicity for NSCLC MHC Class I PresentedPeptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, we performed investigations using an invitro T-cell priming assay based on repeated stimulations of CD8+ Tcells with artificial antigen presenting cells (aAPCs) loaded withpeptide/MHC complexes and anti-CD28 antibody. This way we could showimmunogenicity for 9 HLA-A*0201 restricted TUMAPs of the invention sofar, demonstrating that these peptides are T-cell epitopes against whichCD8+ precursor T cells exist in humans (Table 4).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, we first isolated CD8+ T cells from fresh HLA-A*02leukopheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the Transfusion Medicine Tuebingen, Germany, after informedconsent.

Isolated CD8+ lymphocytes or PBMCs were incubated until use in T-cellmedium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nirnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

All pMHC complexes used for aAPC loading and cytometric readout werederived from UV-induced MHC ligand exchange (Rodenko et al., 2006) withminor modifications. In order to determine the amount of pMHC monomerobtained by exchange we performed streptavidin-based sandwich ELISAsaccording to (Rodenko et al., 2006).

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV from modified Melan-A/MART-1) andA*0201/DDX5-001 (YLLPAIVHI from DDX5), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3-4 days at 37° C. Half of the medium was then exchanged by freshTCM supplemented with 80 U/ml IL-2 and incubating was continued for 3-4days at 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SKI (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+ CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for NSCLC Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for twopeptides of the invention are shown in FIG. 4 together withcorresponding negative controls. Results for 25 peptides from theinvention are summarized in Table 5.

TABLE 5 In vitro immunogenicity of HLA class I peptides of the inventionExemplary results of in vitro immunogenicity experiments conducted bythe applicant for the peptides of the invention. <20% = +; 20%-49% = ++;50%-70% = +++; and >70% = ++++ SEQ ID NO: Wells Donors 1 + ++ 2 + ++ 3 +++ 4 + ++ 7 ++ ++++ 8 + ++ 9 + + 10 + ++ 11 ++ ++++ (100%) 15 ++ ++ 16 +++ 19 + ++ 18 + +++ 21 ++ ++ 22 + +++ 24 + ++ 30 + ++ 31 + +++ 32 + +++33 + +++ 35 + ++ 37 + ++++ (100%) 38 + ++ 39 + ++ 40 + ++ 42 ++ ++++(100%) 43 + +++ 44 + ++ 45 + + 46 + +++ 47 + ++ 48 + + 52 + + 53 ++ ++54 + ++ 55 + ++ 56 ++ ++++ (100%) 62 ++ ++++ 57 + ++ 59 + +++ 60 +++++++ (100%) 61 + +++ 63 + ++ 64 + +++ 65 ++ +++ 66 + +++ 67 + ++ 68 + +69 ++ +++ 70 + +++ 71 + +++ 72 + +++ 73 + ++ 74 + +++ 75 + ++ 78 ++ ++79 + ++++ 80 + ++ 81 + ++ 85 ++ ++++ 86 + ++ 87 + +++ 88 + ++ 92 + ++

Example 5 Syntheses of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. After purification bypreparative RP-HPLC, ion-exchange procedure was performed to incorporatephysiological compatible counter ions (for example trifluoro-acetate,acetate, ammonium or chloride).

Identity and purity of each individual peptide have been determined bymass spectrometry and analytical RP-HPLC. After ion-exchange procedurethe peptides were obtained as white to off-white lyophilizates inpurities of 90% to 99.7%.

All TUMAPs are preferably administered as trifluoro-acetate salts oracetate salts, other salt-forms are also possible. For the measurementsof example 4, trifluoro-acetate salts of the peptides were used.

Example 6 UV-Ligand Exchange

Candidate peptides for the vaccines according to the present inventionwere further tested for immunogenicity by in vitro priming assays. Theindividual peptide-MHC complexes required for these assays were producedby UV-ligand exchange, where a UV-sensitive peptide is cleaved uponUV-irradiation, and exchanged with the peptide of interest as analyzed.Only peptide candidates that can effectively bind and stabilize thepeptide-receptive MHC molecules prevent dissociation of the MHCcomplexes. To determine the yield of the exchange reaction, an ELISA wasperformed based on the detection of the light chain (β2m) of stabilizedMHC complexes. The assay was performed as generally described in Rodenkoet al. (Rodenko B, Toebes M, Hadrup S R, van Esch W J, Molenaar A M,Schumacher T N, Ovaa H. Generation of peptide-MHC class I complexesthrough UV-mediated ligand exchange. Nat Protoc. 2006; 1(3): 1120-32.).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 30min at 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*0201/MLA-001 monomers served as standards, covering the range of8-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.

TABLE 6 UV-Ligand exchange SEQ ID Average exchange NO. Peptide nameyield in % Exchange yield 81 ANKS1A-001 78 ++++ 87 AURKB-001 54 +++ 85BUB1B-001 59 +++ 48 SNRNP20-001 54 +++ 80 CEP192-001 56 +++ 90 COG4-00157 +++ 89 IFT81-001 57 +++ 83 MDN1-001 67 +++ 82 CEP250-002 70 +++ 91NCBP1-001 65 +++ 92 NEFH-001 50 ++ 84 OLFM1-001 48 ++ 86 PI4KA-001 51+++ 11 SLC3A2-001 56 +++ 78 SLI-001 47 ++ 79 TLX3-001 70 +++ 2 MMP12-00357 +++ 68 FAP-003 31 ++ 66 IGF2BP3-001 46 ++ 4 DST-001 50 ++ 5 MXRA5-00157 +++ 31 GFPT2-001 43 ++ 1 ABCA13-001 93 ++++ 6 DST-002 59 +++ 40MXRA5-002 56 +++ 49 SAMSN1-001 47 ++ 8 HNRNPH-001 26 ++ 69 WNT5A-001 37++ 15 IL8-001 41 ++ 50 STAT2-001 69 +++ 72 ADAM8-001 67 +++ 73COL6A3-002 81 ++++ 18 VCAN-001 41 ++ 12 SMYD3-001 50 ++ 3 ABCA13-002 36++ 35 BNC1-001 43 ++ 7 CDK4-001 45 ++ 19 DROSHA-001 68 +++ 33 GALNT2-00173 ++++ 13 AKR-001 13 + 39 LAMC2-001 61 +++ 56 RAD54B-001 48 ++ 24COL12A1-002 55 +++ 43 CSE1-001 55 +++ 45 SEC61G-001 18 + 47 PCNXL3-00187 ++++ 9 TANC2-001 71 ++++ 70 TPX2-001 56 +++ 17 HUWE1-001 45 ++ 54TACC3-001 54 +++ 32 CERC-001 62 +++ 26 SERPINB3-001 47 ++ 58 CCNA2-00154 +++ 44 DPYSL4-001 77 ++++ 27 KIF26B-001 68 +++ 51 CNOT1-001 57 +++ 11SLC34A2-001 51 +++ 30 RGS4-001 49 ++ 20 VCAN-002 49 ++ 67 CDC6-001 48 ++74 THY1-001 65 +++ 10 RNF213-001 84 ++++ 61 RCN1-001 75 ++++ 37 FZD-00152 +++ 71 HMMR-001 49 ++ 60 C11orf24-001 47 ++ 53 JUNB-001 51 +++ 25ELANE-001 62 +++ 61 RCC1-001 77 ++++ 62 MAGEF1-001 83 ++++ 22 ACACA-00161 +++ 21 PLEKHA8-001 47 ++ 57 EEF2-002 31 ++ 41 HSP-002 47 ++ 38ATP-001 19 + 46 ORMDL1-002 61 +++ 59 NET1-001 82 ++++ 63 NCAPD2-001 76++++ 42 VPS13B-001 63 +++ 64 C12orf44-001 34 ++ 23 ITGA11-001 53 +++ 75DIO2-001 50 ++ 28 ANKH-001 52 +++ 65 HERC4-001 61 +++ 16 P2RY6-001 91++++

Candidate peptides that show a high exchange yield (i.e. higher than40%, preferably higher than 50%, more preferred higher than 70%, andmost preferred higher than 80%) are generally preferred for a generationand production of antibodies or fragments thereof, and/or T cellreceptors or fragments thereof, as they show sufficient avidity to theMHC molecules and prevent dissociation of the MHC complexes.

Example 7 Binding and Immunogenicity of Selected MHC Class II Peptides

HLA class II proteins are divided into 3 major isotypes HLA-DR, -DP, DQwhich are encoded by numerous haplotypes. The combination of various α-and β-chains increases the diversity of the HLA class II proteins foundin an arbitrary population. Thus, the selected HLA class II TUMAPs haveto bind to several different HLA-DR molecules (i.e. show promiscuousbinding ability) in order to be able to contribute to an effectiveT-cell response in a significant percentage of patients.

The promiscuous binding of POSTN-002 and MMP12-002 to various HLA-DRhaplotypes and the stability of the formed complexes was assessed in anin vitro binding assay by an external service provider as follows.

Materials and Methods List of Peptides

Sequence Peptide No ID Sequence Origin Size 76 MMP12-002 INNYTPDMNIMA-942 18 REDVDYAIR 77 POSTN-002 TNGVIHVVD IMA-942 17 KLLYPADTList of investigated HLA-DR HaplotypesThe 7 investigated HLA-DR haplotypes are selected according to theirfrequencies in HLA-A*02 and HLA-A*24 positive North Americans population(Table 7.1 and 7.2)

Data are derived from the analysis of 1.35 million HLA-typed volunteersregistered in the National Marrow Donor Program (Mori et al., 1997). Theanalyzed population was subdivided in the following ethnic groups:Caucasian Americans (N=997,193), African Americans (N=110,057), AsianAmericans (N=81,139), Latin Americans (N=100,128), and Native Americans(N=19,203).

Principle of Test

The ProImmune REVEAL® MHC-peptide binding assay determines the abilityof each candidate peptide to bind to the selected HLA class II haplotypeand stabilize the HLA-peptide complex. Thereby the candidate peptidesare assembled in vitro with a particular HLA class II protein. The levelof peptide incorporation into HLA molecules is measured by presence orabsence of the native conformation of the assembled HLA-peptide complexat time 0 after completed refolding procedure (so called on-rate).

The binding capacity of candidate peptide to a particular HLA moleculeis compared to the one with known very strong binding properties(positive control) resulting in the corresponding REVEAL® MHC-peptidebinding score. The positive control peptide is selected and provided byProImmune based on their experience individually for each HLA haplotype.

Besides the affinity of a peptide to a particular HLA molecule, theenduring stability of the formed HLA-peptide complex is crucial for theoccurrence of an immune response. Accordingly presence of the formedHLA-peptide complex is measured after its incubation for 24 h at 37° C.Consequently the stability of the formed MHC-peptide complex iscalculated as a ration of the binding scores at 24 h and the bindingscores which are received right after the refolding (accordingly at time0) in percent.

Results

The analysis of POSTN-002 and MMP12-002 in REVEAL® MHC-peptide bindingassay showed that both peptides bind to various HLA haplotypes.POSTN-002 was shown to form a complex with 5 and MMP12-002 with 4 of 7investigated HLA haplotypes (FIG. 5). Both peptides did not bind toHLA-DR3 and HLA-DR6. The detected binding scores were within the rangeof 0.02 to about 2.5% compared to the positive control, and clearlyabove scores of non-binding peptides.

The stability analysis of the formed HLA-POSTN-002 and HLA-MMP12-002complexes revealed that 3 and 2 of 6 investigated HLA-peptide complexeswere stable after 24 h at 37° C., respectively (FIG. 6).

A conclusion on the immugenicity of a peptide based on its bindingcapacity to a HLA molecule can be made by comparing the binding score ofthis peptide to the one with known immunogenicity. Therefore, five wellinvestigated peptides with determined immunogenicity were selected forthis comparison. The immunogenicity of these peptides was determined exvivo in blood samples of vaccinated patients using intracellularcytokine staining (ICS) CD4 T-cells.

In principle, ICS assays analyze the quality of specific T cells interms of effector functions. Therefore, the peripheral mononuclear cells(PBMCs) were cultivated in vitro and subsequently restimulated by thepeptide of interest, a reference peptide and a negative control (hereMOCK). Following the restimulated cells were stained for FN-gamma,TNF-alpha, IL-2 and IL-10 production, as well as expression of theco-stimulatory molecule CD154. The counting of affected cells wasperformed on a flow cytometer (FIG. 7).

The immunogenicity analysis revealed 100% immune response by vaccinationwith IMA950 peptides (BIR-002 and MET-005) in 16 patients and 44% to 86%immune response by vaccination with IMA910 peptides (CEA-006, TGFBI-004and MMP-001) in 71 patients.

To compare the binding scores of POSTN-002 and MMP12-002 to the bindingscores of IMA910 and IMA950 peptides, all peptides were arranged in atable for each investigated HLA-DR haplotype according to the detectedbinding score (Tables 8.1 to 8.5).

The comparison of the binding scores of POSTN-002 and MMP12-002 to thebinding scores of the other class II peptides with known immunogenicityshowed that the binding capacities of both peptides are mostly locatedin the middle till the lower half of the tables with exception ofHLA-DR2. The binding capacities of both peptides to HLA-DR2 are locatedin the upper half of the table with MMP12-002 being the top candidate.Based on this analysis it must be expected that both peptides, POSTN-002and MMP12-002, induce an immune response as well.

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1. A peptide comprising an amino acid sequence of SEQ ID 2, or a variantsequence thereof which is at least 80% homologous to SEQ ID No. 2,wherein said variant induces antibodies and/or T cells specificallybinding to said peptide, or a pharmaceutical acceptable salt of SEQ IDNo. 2, wherein said peptide is not the underlying full-lengthpolypeptide.
 2. The peptide according to claim 1, wherein said peptideor variant thereof has an overall length of between 8 and 100,optionally between 8 and 30, of optionally between 8 and 14 amino acidsfor a peptide comprising SEQ ID No.
 2. 3. The peptide according claim 1,having the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.
 4. The peptideaccording claim 1, wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No.
 2. 5. The peptideaccording to claim 1, wherein said peptide is modified, elongated N-and/or C-terminally by up to four amino acids, and/or includesnon-peptide bonds.
 6. The peptide according to claim 1, wherein saidpeptide is a part of a fusion protein, optionally fused to theN-terminal amino acids of the HLA-DR antigen-associated invariant chain(Ii), or is fused to an antibody.
 7. A nucleic acid, encoding for apeptide according to claim
 1. 8. The nucleic acid according to claim 7,which is DNA, cDNA, PNA, RNA or combinations thereof.
 9. An expressionvector capable of expressing the nucleic acid according to claim
 7. 10.The peptide according to claim 1 capable for use in medicine.
 11. A hostcell, comprising the nucleic acid according to claim 7, wherein saidhost cell is not a human embryonic stem cell.
 12. The host cellaccording to claim 11, wherein said cell is an antigen presenting cell,optionally a dendritic cell.
 13. A pharmaceutical composition comprisingthe peptide according to claim 1 or a pharmaceutical acceptable saltthereof, and at least one other component selected from the group ofpharmaceutically acceptable, optionally aqueous, carriers and/orexcipients, optionally buffers, binding agents, blasting agents,diluents, flavours, lubricants, and immune stimulating or immunemodulating substances, such as cytokines, immuno-modulators, adjuvantsand therapeutic substances with immune modulating properties.
 14. Methodfor producing a peptide the method comprising culturing the host cellaccording to claim 11, and isolating said peptide from the host cell orculture medium thereof.
 15. An in vitro method for producing anactivated cytotoxic T lymphocyte (CTL) or T helper cell (Th cell), themethod comprising contacting in vitro a CTL or Th cell withantigen-loaded human class I or II MHC molecules expressed on thesurface of a suitable antigen-presenting cell for a period of timesufficient to activate said CTL in an antigen specific manner, whereinsaid antigen is the peptide according to claim
 1. 16. The methodaccording to claim 15, wherein said antigen is loaded onto class I or IIMHC molecules expressed on the surface of a suitable antigen-presentingcell by contacting a sufficient amount of said antigen with saidantigen-presenting cell.
 17. The method according to claim 151, whereinsaid antigen-presenting cell comprises an expression vector capable ofexpressing a peptide according to claim
 1. 18. An activated cytotoxic Tlymphocyte (CTL) or T helper cell (Th cell), produced by the methodaccording to claim 15, wherein said CTL or Th cell selectivelyrecognises a cell which aberrantly expresses a polypeptide comprising anamino acid sequence as given in said peptide.
 19. An in vitro method forproducing a TCR or sTCR or fragment thereof that is specific for thepeptide according to claim 1, comprising cloning of variable domainsfrom an activated cytotoxic T lymphocyte (CTL) or T helper cell (Thcell), and expressing said TCR or sTCR or fragment thereof in a suitablehost and/or expression system.
 20. An isolated binding agent optionallyan antibody or fragment thereof, a protein, a nucleic acid, a peptide, aTCR or an sTCR or fragment thereof that binds, and optionally bindsspecifically, to the peptide according to claim 1 or to a complex of thepeptide.
 21. An isolated T-cell receptor reactive with an HLA ligandthat is at least 80% identical with an amino acid sequence of SEQ ID 2.22. The isolated T-cell receptor of claim 21, wherein said amino acidsequence is at least 90%, optionally at least 95%, optionally 100%identical to SEQ ID No.
 2. 23. A method of killing target cells in apatient, wherein said target cells aberrantly express and/or present apolypeptide comprising an amino acid sequence as given in claim 1, themethod comprising administering to said patient an effective number ofcytotoxic T lymphocytes (CTL) or Th cells.
 24. A peptide according toclaim 1 as a medicament, wherein said medicament is active againstcancer.
 25. The peptide according to claim 24, wherein said medicamentis a vaccine.
 26. The peptide according to claim 24, wherein said canceris selected from non-small cell lung carcinoma (NSCLC), lung cancer,gastric cancer, and/or glioblastoma.
 27. The peptide according to claim24 for adoptive cellular therapy in humans.
 28. An autologous orallogeneic human cytotoxic T cell (CTL) or T helper cell (Th cell),recombinantly transfected with an isolated binding agent optionally aT-cell receptor according to claim
 20. 29. A pharmaceutical compositioncomprising: (a) an entity selected from the group consisting of: (a1) anisolated peptide according to claim 1, (a2) a T cell receptor, sTCR orfragment thereof (a3) a fusion protein (a4) a nucleic acid (a5) anexpression vector (a6) a host cell (a7) an activated cytotoxic Tlymphocyte or T helper cell wherein a2-a7 comprise or are derived fromsaid peptide, and (b) a pharmaceutically acceptable carrier, andoptionally (c) at least one other component selected from the group ofpharmaceutically acceptable excipients, buffers, binding agents,blasting agents, diluents, flavours, lubricants, and immune stimulatingor immune modulating substances, such as cytokines, immunomodulators,adjuvants and therapeutic substances with immune modulating properties